Natural combination hormone replacement formulations and therapies

ABSTRACT

Pharmaceutical formulations for co-administering estradiol and progesterone are provided herein. In some embodiments, the formulation comprises solubilized estradiol, suspended progesterone, and a medium chain (C6-C12) oil.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/843,428, filed on Mar. 15, 2013, which is acontinuation-in-part of U.S. application Ser. No. 13/684,002, filed onNov. 21, 2012, which claims priority to U.S. Provisional ApplicationSer. No. 61/661,302, filed on Jun. 18, 2012, and to U.S. ProvisionalApplication Ser. No. 61/662,265, filed on Jun. 20, 2012; and also claimspriority to U.S. Provisional Application Ser. No. 61/889,483, filed Oct.10, 2013; the contents of each of which are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

This application relates to natural estrogen and progesteronereplacement therapies, with formulations provided for each estradiol andprogesterone alone and in combination for the treatment of pre,peri-menopausal, menopausal and post-menopausal females in relation tothe treatment of Estrogen- and Progesterone-deficient states, each asherein below defined.

BACKGROUND OF THE INVENTION

Hormone Replacement Therapy (HRT) is a medical treatment that involvesthe use of one or more of a group of medications designed to increasehormone levels in women who lack adequate hormone production. HRT canmitigate and prevent symptoms caused by diminished circulating estrogenand progesterone hormones regardless as to whether the subject ispre-menopausal, peri-menopausal, menopausal or post-menopausal. However,specific symptomatic states can exist during each stage of menopausalprogression.

HRT is presently available in various forms. One therapy involvesadministration of low dosages of one or more estrogens. Another involvesadministration of progesterone or a chemical analogue, called aprogestin. Progesterone administration acts, among treating otherdisease states, to mitigate certain undesirable side effects fromestrogen administration including, for example, endometrial hyperplasia(thickening) and reducing the incidence of endometrial cancer.

Timing for dosage administration is often varied cyclically, withestrogens taken daily and progesterone taken for approximately two weeksof every month; a method often referred to as “Cyclic-Sequential” or“Sequentially-Combined HRT.” This method is intended to mimic thenatural menstrual cycle and typically causes menstruation similar to aperiod after the progesterone is stopped. This regimen is most typicallyused in peri-menopausal or newly menopausal women as the alternativecontinuous method often results in irregular bleeding in such women. Analternate method, a constant dosage with both estrogen and progesteronetaken daily, is called “Continuous-Combined HRT.” This method usuallyresults in no menstruation and is used most often after a woman has beenmenopausal for some time.

Estrogen, in its various forms, and progesterone, in its various forms,are used in HRT via a variety of administered dosage forms including,for example, via tablets, capsules and patches.

“Bio-identical” or “body-identical” hormones, which are identical inchemical structure to the hormones naturally produced by human bodies,can be used and are often referred to as Natural Hormone ReplacementTherapy, or NHRT.

These natural or bio-identical hormones are formulated from variousingredients to match the chemical structure and effect of estradiol,estrone, or estriol (the 3 primary estrogens) as well as progesteronethat occurs naturally in the human body (endogenous).

Currently, bio-identical estradiol is available in both branded andgeneric FDA approved versions. FDA-approved bio-identical progesteronefor HRT is available as the branded stand-alone drug commerciallyidentified as PROMETRIUM® (progesterone, USP) (Abbott Laboratories,Abbott Park, Ill.), with a generic authorized by the innovator, andgeneric products provided by Teva (Israel) and Sofgen Americas, Inc.(New York). PROMETRIUM® was approved for sale in the United States onMay 14, 1998 under NDA # N019781. According to the prescribinginformation approved for this product (Rev June 2009) (“PROMETRIUM®prescribing information”), PROMETRIUM® comprises synthetic progesteronethat is chemically identical to progesterone of human ovarian origin.Capsules comprise 100 mg or 200 mg of micronized progesterone. Theinactive ingredients include peanut oil, gelatin, glycerin, lecithin,titanium dioxide, and yellow and red dyes.

Other products such as PREMPRO® (conjugatedestrogens/medroxyprogesterone acetate tablets) and PREMPHASE®(conjugated estrogens plus medroxyprogesterone acetate tablets) (WyethLaboratories, a division of Pfizer, Inc., New York) provide bothcontinuous-combined and cyclic-sequential products containing PREMARIN®(estrogen derived from mare's urine) and synthetic medroxyprogesteroneacetate. Other products are available. However, no FDA approved productexists on the market today with combination bio-identical estradiol andbio-identical progesterone.

BRIEF SUMMARY OF THE INVENTION

In one aspect, pharmaceutical formulations for co-administeringestradiol and progesterone to a mammal in need thereof are provided. Insome embodiments, the pharmaceutical formulation comprises: solubilizedestradiol, suspended progesterone, and a medium chain (C6 to C12) oil.

In some embodiments, the medium chain oil comprises medium chain fattyacid esters of glycerol, polyethylene glycol, or propylene glycol, ormixtures thereof, wherein the medium chain fatty acids arepredominantly: C6 to C12 fatty acids, C6 to C10 fatty acids, C8 to C12fatty acids, or C8 to C10 fatty acids. In some embodiments, the mediumchain oil comprises a glyceride containing a C6-C12 fatty acid. In someembodiments, the glyceride is a mixture of mono- and diglycerides. Insome embodiments, the fatty acid is predominantly a C8 to C10 fattyacid. In some embodiments, the fatty acids are predominantly saturatedfatty acids. In some embodiments, the fatty acids are predominantlyunsaturated fatty acids. In some embodiments, the medium chain oilcomprises CAPMUL® MCM.

In some embodiments, the pharmaceutical formulation further comprisesone or more surfactants, e.g., one or more non-ionic surfactants. Insome embodiments, the surfactant comprises lauroylpolyoxyl-32-glycerides. In some embodiments, the surfactant comprisesGELUCIRE® 44/14.

In some embodiments, the pharmaceutical formulation further comprisessolubilized progesterone, wherein at least 50% (e.g., at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, or about 100%) ofthe total progesterone is solubilized.

In some embodiments, the pharmaceutical formulation is in a hardcapsule, a soft capsule, or a tablet for oral administration. In someembodiments, the hard capsule or soft capsule comprises gelatin,glycerol, and coloring agents.

In some embodiments, the pharmaceutical formulation comprises:

30 to 35 wt % progesterone;

0.1 to 0.4 wt % estradiol;

55 to 75 wt % medium chain oil; and

0.5 to 10 wt % non-ionic surfactant.

In some embodiments, the pharmaceutical formulation comprises about 2 mgestradiol and about 200 mg progesterone.

In some embodiments, a pharmaceutical formulation as described herein(e.g., comprising solubilized estradiol, suspended progesterone, and amedium chain (C6-C12) oil), when administered to a human subject,produces:

(a) one or more progesterone-related parameters selected from: (i) anarea under the curve (AUC)_((0-t)) for progesterone that is from 96ng·hr/ml to 150 ng·hr/ml; (ii) an AUC_((0-∞)) for progesterone that isfrom 105 ng·hr/ml to 164 ng·hr/ml; and (iii) a C_(max) for progesteronethat is from 71 ng/ml to 112 ng/ml; and

(b) one or more estrogen-related parameters selected from: (i) anAUC_((0-t)) for estradiol that is from 1123 pg·hr/ml to 1755 pg·hr/ml;(ii) an AUC_((0-∞)) for estradiol that is from 1968 pg·hr/ml to 3075pg·hr/ml; and (iii) a C_(max) for unconjugated estradiol that is from 52pg/ml to 81 pg/ml.

In some embodiments, administration of the formulation to the subjectproduces both an AUC_((0-t)) for progesterone that is from 96 ng·hr/mlto 150 ng·hr/ml and a C_(max) for progesterone that is from 71 ng/ml to112 ng/ml. In some embodiments, administration of the formulation to thesubject produces both an AUC_((0-t)) for unconjugated estradiol that isfrom 1123 pg·hr/ml to 1755 pg·hr/ml and a C_(max) for unconjugatedestradiol that is from 52 pg/ml to 81 pg/ml.

In some embodiments, when the formulation is administered to a humansubject, the formulation further produces one or more of the following:

(i) an AUC_((0-t)) for estrone sulfate that is from 7277 pg·hr/ml to11370 pg·hr/ml;

(ii) an AUC_((0-∞)) for estrone sulfate that is from 9596 pg·hr/ml to14994 pg·hr/ml; or

(iii) a C_(max) for estrone sulfate that is from 341 pg/ml to 533 pg/ml.

In some embodiments, administration of the formulation to the subjectproduces both an AUC_((0-t)) for estrone sulfate that is from 7277pg·hr/ml to 11370 pg·hr/ml and a C_(max) for estrone sulfate that isfrom 341 pg/ml to 533 pg/ml.

In some embodiments, when the formulation is administered to a humansubject, the formulation further produces one or more of the following:

(i) an AUC_((0-t)) for total estrone that is from 161 pg·hr/ml to 252pg·hr/ml;

(ii) an AUC_((0-∞)) for total estrone that is from 171 pg·hr/ml to 267pg·hr/ml; or

(iii) a C_(max) for total estrone that is from 28 pg/ml to 44 pg/ml.

In some embodiments, administration of the formulation to the subjectproduces both an AUC_((0-t)) for total estrone that is from 161 pg·hr/mlto 252 pg·hr/ml and a C_(max) for total estrone that is from 28 pg/ml to44 pg/ml.

In some embodiments, the progesterone and the estradiol in thepharmaceutical formulation demonstrate comparable bioavailability totheir individual drug references of PROMETRIUM® and ESTRACE®,respectively (“Referenced Products”), when said formulation isadministered to a human subject.

In some embodiments, the progesterone and the estradiol in saidformulation demonstrate about 80% to about 125% of the C_(max) and/orAUC of their individual references of PROMETRIUM® and ESTRACE®,respectively, when said formulation is administered to a human subject.

In some embodiments, when administered to a human subject, theformulation produces one or more of the following:

(i) an AUC_((0-t)) for progesterone comparable to the AUC_((0-t)) forprogesterone obtained with PROMETRIUM®;

(ii) an AUC_((0-∞)) for progesterone comparable to the AUC_((0-∞)) forprogesterone obtained with PROMETRIUM®; or

(iii) a C_(max) for progesterone comparable to the C_(max) forprogesterone obtained with PROMETRIUM®.

In some embodiments, when administered to a human subject, theformulation produces one or more of the following:

(i) an AUC_((0-t)) for unconjugated estradiol comparable to theAUC_((0-t)) for progesterone obtained with ESTRACE®;

(ii) an AUC_((0-∞)) for unconjugated estradiol comparable to theAUC_((0-∞)) for progesterone obtained with ESTRACE®; or

(iii) a C_(max) for unconjugated estradiol comparable to the C_(max) forprogesterone obtained with ESTRACE®.

In some embodiments, when administered to a human subject, theformulation produces one or more of the following:

(i) an AUC_((0-t)) for unconjugated estrone comparable to theAUC_((0-t)) for progesterone obtained with ESTRACE®;

(ii) an AUC_((0-∞)) for unconjugated estrone comparable to theAUC_((0-∞)) for progesterone obtained with ESTRACE®; or

(iii) a C_(max) for unconjugated estrone comparable to the C_(max) forprogesterone obtained with ESTRACE®.

In some embodiments, when administered to a human subject, theformulation produces one or more of the following:

(i) an AUC_((0-t)) for total estrone comparable to the AUC_((0-t)) forprogesterone obtained with ESTRACE®; or

(ii) an AUC_((0-∞)) for total estrone comparable to the AUC_((0-∞)) forprogesterone obtained with ESTRACE®.

In another aspect, methods of treating a subject having one or moresymptoms of estrogen deficiency (e.g., one or more symptoms ofmenopause) are provided. In some embodiments, the method comprisingadministering to the subject an effective amount of a pharmaceuticalformulation as described herein. In some embodiments, the subject isfemale. In some embodiments, the subject is a woman having a uterus.

In yet another aspect, methods of effecting hormone replacement therapyin a woman in need thereof are provided. In some embodiments, the methodcomprises orally administering to the woman an effective amount of apharmaceutical formulation as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate the present disclosure and, togetherwith the description, further serve to explain the principles of thedisclosure and to enable a person skilled in the pertinent art to makeand use the disclosed embodiments.

FIG. 1 illustrates an exemplary manufacturing process of a fill materialin accordance with various embodiments of the invention;

FIG. 2 illustrates an exemplary manufacturing process of a softgelmaterial in accordance with various embodiments of the invention;

FIG. 3 illustrates an exemplary manufacturing process in accordance withvarious embodiments of the invention;

FIG. 4 illustrates a graph of the particle distribution obtained inExample 10; and

FIG. 5 illustrates a dissolution study of a formulation in accordancewith various embodiments of the invention.

FIG. 6 illustrates a flow chart of subject disposition for a studycomparing the bioavailability of a 17β-estradiol and progesteronecombination formulation to the bioavailability of co-administeredPROMETRIUM® and ESTRACE®.

FIG. 7 illustrates pharmacokinetic parameters for progesterone for thecombination formulation (T) versus co-administered PROMETRIUM® andESTRACE® (R1 and R2).

FIG. 8 illustrates pharmacokinetic parameters for free estradiol for thecombination formulation (T) versus co-administered PROMETRIUM® andESTRACE® (R1 and R2).

DETAILED DESCRIPTION OF THE INVENTION

Frequently, higher recommended oral dosages of pharmaceuticals arenecessary to treat a given disease state because many active ingredientsare not completely absorbed by a patient in need of treatment. In otherwords, a better-absorbed dosage form of a medicament such as, forexample, progesterone or estradiol, or dosage forms that provide greaterconsistency of absorption of progesterone or estradiol among subjects,alone or in combination with estradiol, may be able to be administeredat dosage strengths lower than presently recommended, potentiallyresulting in a reduced or minimized side effect profile, among otherpotential benefits.

I. DEFINITIONS

The term “area under the curve” (“AUC”) refers to the area under thecurve defined by changes in the blood concentration of an activepharmaceutical ingredient (e.g., estradiol or progesterone), or ametabolite of the active pharmaceutical ingredient, over time followingthe administration of a dose of the active pharmaceutical ingredient.“AUC_(0-∞)” is the area under the concentration-time curve extrapolatedto infinity following the administration of a dose. “AUC_(0-t)” is thearea under the concentration-time curve from time zero to time tfollowing the administration of a dose, wherein t is the last time pointwith measurable concentration.

The term “C_(max)” refers to the maximum value of blood concentrationshown on the curve that represents changes in blood concentrations of anactive pharmaceutical ingredient (e.g., progesterone or estradiol), or ametabolite of the active pharmaceutical ingredient, over time.

The term “T_(max)” refers to the time that it takes for the bloodconcentration an active pharmaceutical ingredient (e.g., estradiol orprogesterone), or a metabolite of the active pharmaceutical ingredient,to reach the maximum value.

Collectively AUC, C_(max) and, optionally, T_(max) are the principalpharmacokinetic parameters that can characterize the pharmacokineticresponse of a particular drug product, such as progesterone orestradiol, in an animal, especially a mammal, including human, subject.

An “active pharmaceutical ingredient” (API), as used herein, means theactive compound or compounds used in formulating a drug product. APIsare generally safe for administering to animals, especially mammals,including humans, according to established governmental standards,including those promulgated by the United States Food and DrugAdministration.

The term “bioavailability” has the meaning as defined in 21 C.F.R.§320.1(a): the rate and extent to which an API or active ingredient oractive moiety is absorbed from a drug product and becomes available atthe site of action. For drug products that are not intended to beabsorbed into the bloodstream, bioavailability may be assessed bymeasurements intended to reflect the rate and extent to which the API oractive ingredient or active moiety becomes available at the site ofaction. For example, bioavailability can be measured as the amount ofAPI in the blood (serum or plasma) as a function of time.Pharmacokinetic (PK) parameters such as AUC, C_(max), or T_(max) may beused to measure and assess bioavailability.

The term “bioequivalent” has the meaning as defined in 21 C.F.R.§320.1(e): the absence of a significant difference in the rate andextent to which the API or active ingredient or active moiety inpharmaceutical equivalents or pharmaceutical alternatives becomesavailable at the site of drug action when administered at the same molardose under similar conditions in an appropriately designed study. Wherethere is an intentional difference in rate (e.g., in certain extendedrelease dosage forms), certain pharmaceutical equivalents oralternatives may be considered bioequivalent if there is no significantdifference in the extent to which the active ingredient or moiety fromeach product becomes available at the site of drug action. This appliesonly if the difference in the rate at which the active ingredient ormoiety becomes available at the site of drug action is intentional andis reflected in the proposed labeling, is not essential to theattainment of effective body drug concentrations on chronic use, and isconsidered medically insignificant for the drug. In practice, twoproducts are considered bioequivalent if the 90% confidence interval ofthe AUC, C_(max), or optionally T_(max) is within 80.00% to 125.00%.

As used herein, the term “comparable,” as used with reference tocomparing a bioavailability characteristic (including but not limited toarea under the curve (AUC), C_(max), or T_(max)) for a test composition,means that the test composition has a value for the bioavailabilitycharacteristic that is from 80% to 125% of the value of thebioavailability characteristic of a reference composition. In someembodiments, the reference composition is a commercially availableprogesterone composition (e.g., progesterone in a peanut oil, e.g.,PROMETRIUM®) or a commercially available estradiol composition (e.g., amicronized estradiol tablet, e.g., ESTRACE®). In some embodiments, thereference composition is a co-administration of a commercially availableprogesterone composition (e.g., PROMETRIUM®) and a commerciallyavailable estradiol composition (e.g., ESTRACE®). In some embodiments,the reference composition is a combination formulation comprisingprogesterone and estradiol as provided herein. Thus, in someembodiments, a test composition is “comparable” to a referencecombination formulation comprising progesterone and estradiol asprovided herein when the test composition has a value for abioavailability characteristic (e.g., AUC_((0-t)), AUC_((0-∞)), C_(max),and/or T_(max) for one or more analytes, e.g., progesterone,unconjugated estradiol, unconjugated estrone, or total estrone) that isfrom 80% to 125% of the value of the bioavailability characteristic ofthe reference combination formulation.

The term “bio-identical hormone” or “body-identical hormone” refers toan active pharmaceutical ingredient that is structurally identical to ahormone naturally or endogenously found in the human body (e.g.,estradiol and progesterone).

The term “estradiol” refers to (17β)-estra-1,3,5(10)-triene-3,17-diol.Estradiol is also interchangeably called 17β-estradiol, oestradiol, orE2, and is found endogenously in the human body. As used herein,estradiol refers to the bio-identical or body-identical form ofestradiol found in the human body having the structure:

As used herein, unless specified, estradiol includes estradiol inanhydrous and hemihydrate forms. For the purposes of this disclosure,the anhydrous form or the hemihydrate form can be substituted for theother by accounting for the water or lack of water according towell-known and understood techniques.

The term “solubilized estradiol” means that the estradiol or a portionthereof is solubilized or dissolved in the solubilizing agents or theformulations disclosed herein. Solubilized estradiol may includeestradiol that is about 80% solubilized, about 85% solubilized, about90% solubilized, about 95% solubilized, about 96% solubilized, about 97%solubilized, about 98% solubilized, about 99% solubilized or about 100%solubilized. In some embodiments, the estradiol is “fully solubilized”with all or substantially all of the estradiol being solubilized ordissolved in the solubilizing agent. Fully solubilized estradiol mayinclude estradiol that is about 97% solubilized, about 98% solubilized,about 99% solubilized or about 100% solubilized. Solubility can beexpressed as a mass fraction (% w/w, which is also referred to as wt %).

The term “progesterone” refers to pregn-4-ene-3,20-dione. Progesteroneis also interchangeably called P4 and is found endogenously in the humanbody. As used herein, prostergone refers to the bio-identical orbody-identical form of progesterone found in the human body having thestructure:

The term “solubilized progesterone” means that the progesterone or aportion thereof is solubilized or dissolved in the solubilizing agentsor the formulations disclosed herein disclosed herein. In someembodiments, the progesterone is “partially solubilized” with a portionof the progesterone being solubilized or dissolved in the solubilizingagent and a portion of the progesterone being suspended in thesolubilizing agent. The term “partially solubilized progesterone,” asused herein, means progesterone which is in any state of solubilizationup to but not including about 100%, e.g., about 1% solubilized, about 5%solubilized, about 10% solubilized, about 15% solubilized, about 20%solubilized, about 30% solubilized, about 40% solubilized, about 50%solubilized, about 60% solubilized, about 70% solubilized, about 80%solubilized, about 85% solubilized, about 90% solubilized, about 95%solubilized, or about 98% solubilized. In other embodiments, theprogesterone is “fully solubilized” with all or substantially all of theprogesterone being solubilized or dissolved in the solubilizing agent,e.g., at least about 98% solubilized, about 99% solubilized or about100% solubilized. Solubility can be expressed as a mass fraction (% w/w,which is also referred to as wt %).

The terms “micronized progesterone” and “micronized estradiol,” as usedherein, include micronized progesterone and micronized estradiol,respectively, having an X50 particle size value below about 15 micronsand/or having an X90 particle size value below about 25 microns. Theterm “X50,” as used herein, means that one-half of the particles in asample are smaller in diameter than a given number. For example,micronized progesterone having an X50 of 5 microns means that, for agiven sample of micronized progesterone, one-half of the particles havea diameter of less than 5 microns. Similarly, the term “X90” means thatninety percent (90%) of the particles in a sample are smaller indiameter than a given number.

The terms “uniform distribution,” “uniform dispersal,” and “uniformlydispersed,” as used with reference to estradiol or progesterone, meansat least one of uniform dispersion, solubility, or lack of agglomerationof estradiol or progesterone in a dissolution test compared to areference product (e.g., PROMETRIUM® or ESTRACE®, respectively) at asimilar dosage strength and the same USP dissolution apparatus.

The terms “solubilizer” and “solubilizing agent” refer to any substanceor mixture of substances that may be used to solubilize or to enhancethe solubility of an active pharmaceutical ingredient (e.g., estradiolor progesterone). For example and without limitation, suitablesolubilizing agents include medium chain oils and other solvents andco-solvents (e.g., surfactants) that solubilize or dissolve an activepharmaceutical ingredient to a desirable extent. Solubilizing agentssuitable for use in the formulations disclosed herein are pharmaceuticalgrade solubilizing agents (e.g., pharmaceutical grade medium chainoils). It will be understood by those of skill in the art that otherexcipients or components can be added to or mixed with the solubilizingagent to enhance the properties or performance of the solubilizing agentor resulting formulation. Examples of such excipients include, but arenot limited to, surfactants, emulsifiers, thickeners, colorants,flavoring agents, etc. In some embodiments, the solubilizer orsolubilizing agent is a medium chain oil and, in some other embodiments,the medium chain oil is combined with a co-solvent(s) or otherexcipient(s).

The term “medium chain” is used to describe the aliphatic chain lengthof fatty acid containing molecules. As used herein, “medium chain” meansany medium chain carbon-containing substance, including C4-C18, andincluding C6-C12 substances, fatty acid esters of glycerol, fatty acids,and mono-, di-, and tri-glycerides of such substances. In someembodiments, “medium chain” refers to fatty acids, fatty acid esters, orfatty acid derivatives that contain fatty acid aliphatic tails or carbonchains that contain between 6 (C6) and 14 (C14) carbon atoms. Asnon-limiting examples, C6-C14 fatty acids, C6-C12 fatty acids, andC8-C10 fatty acids are all medium chain fatty acids and may be used ininstances in which this specification calls for use of medium chainfatty acids, e.g., medium chain fatty acid esters of glycerol or otherglycols. Examples include, without limitation, caproic acid, caprylicacid, capric acid, lauric acid, myristic acid, and derivatives thereof.

The term “oil,” as used herein, may be any pharmaceutically acceptableoil, such as an organic oil other than peanut oil, that would suspendand/or solubilize any suitable progesterone or estradiol, startingmaterial, or precursor, including micronized progesterone or estradiolas described herein. In some embodiments, oils may include, for exampleand without limitation, medium chain fatty acids, generally of the groupknown as medium chain fatty acids consisting of at least one mono-, di-,or triglyceride, or derivatives thereof, or combinations thereof.

The term “medium chain oil” refers to an oil wherein the composition ofthe fatty acid fraction of the oil is substantially or predominantlymedium chain (e.g., C6 to C14) fatty acids, i.e., the compositionprofile of fatty acids in the oil is substantially medium chain. As usedherein, “substantially” or “predominantly” means that between 20% and100% (inclusive of the upper and lower limits) of the fatty acidfraction of the oil is made up of medium chain fatty acids. In someembodiments, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, or about 95% of the fatty acid fraction ofthe oil is made up of medium chain fatty acids. It will be understood bythose of skill in the art that the medium chain oils suitable for use inthe formulations disclosed herein are pharmaceutical grade (e.g.,pharmaceutical grace medium chain oils). Examples of medium chain oilsinclude, for example and without limitation, medium chain fatty acids,medium chain fatty acid esters of glycerol (e.g., for example, mono-,di-, and triglycerides), medium chain fatty acid esters of propyleneglycol, medium chain fatty acid derivatives of polyethylene glycol, andcombinations thereof.

II. FORMULATIONS

In one aspect, this disclosure relates to pharmaceutical formulationsfor co-administering estradiol and progesterone to a human subject inneed thereof. In some embodiments, the formulation comprises estradiol,progesterone, and a medium chain oil (e.g., a C6-C12 oil). In someembodiments, a pharmaceutical formulation comprising progesterone andestradiol as described herein demonstrates comparable bioavailability totheir individual drug references of PROMETRIUM® and ESTRACE®,respectively, when the formulation is administered to a human subject ora population of subjects.

Another aspect of the present disclosure includes a pharmaceuticalformulation of micronized progesterone, micronized progesterone withpartially solubilized progesterone and fully solubilized progesterone,wherein the formulation may provide increased progesteronebioavailability in a treated subject compared to the bioavailabilityprovided by PROMETRIUM® when administered at equal dosage strengths.

Additional objects of the present disclosure includes: providingincreased patient compliance secondary to ease of use; providingincreased physician adoption secondary to ease of use/instruction withless worry of side effects from inappropriate usage; providing decreasedside-effects from erroneous use (decreased irregular bleeding);providing better efficacy/control of symptoms secondary to appropriateuse; reducing the metabolic and vascular side effects of the commonlyused synthetic progestins when administered alone or in combination withan estrogen (norethindrone acetate, medroxyprogesterone acetate, etc.)including, for example, stroke, heart attacks, blood clots and breastcancer.

Formulations of Estradiol and Progesterone

In some embodiments, a pharmaceutical formulation for use as describedherein comprises solubilized estradiol without progesterone; micronizedprogesterone without estradiol; micronized progesterone with partiallysolubilized progesterone; solubilized estradiol with micronizedprogesterone; solubilized estradiol with micronized progesterone incombination with partially solubilized progesterone; or solubilizedestradiol with solubilized progesterone. The underlying formulationconcepts provided herein may be used with other natural or syntheticforms of estradiol and progesterone. Unless otherwise specified,“natural,” as used herein with reference to hormones discussed herein,means bio-identical or body-identical hormones formulated to match thechemical structure and effect of those that occur naturally in the humanbody (endogenous). An exemplary natural estrogen is estradiol (alsodescribed as 17β-estradiol and E2) and a natural progestin isprogesterone. Micronization specifications, aspects and embodiments arefurther defined herein.

Other aspects of the present disclosure further provide: more uniformdissolution of progesterone, and reduced intra- and inter-patient bloodlevel variability in formulations of progesterone of the presentdisclosure, typically in combinations with solubilized estradiol, whencompared to equal dosages of PROMETRIUM®). Blood level variability isalso compared at equal sampling times following administration. Not tobe limited by theory, these aspects are believed to be influenced by thepercentage of solubilized progesterone in a respective formulationwherein such more uniform dissolution of progesterone, and lower intra-and inter-patient blood level variability, are influenced by a greaterproportion of solubilized progesterone relative to total progesterone. Areduced food effect with the present formulations comprisingprogesterone may also be implicated.

According to the PROMETRIUM® prescribing information, clinical trialshave shown significant patient variability. For example, a clinicaltrial involving post-menopausal women who were administered PROMETRIUM®once a day for five days resulted in the mean PK parameters listed inthe following table:

TABLE 1 PROMETRIUM Capsules Daily Dose Parameter 100 mg 200 mg 300 mgC_(max) (ng/ml) 17.3 +/− 21.9  38.1 +/− 37.8 60.6 +/− 72.5 T_(max) (hr) 1.5 +/− 0.8   2.3 +/− 1.4  1.7 +/− 0.6 AUC₀₋₁₀ (ngxhr/ml) 43.4+/− 30.8101.2 +/− 66.0 175.7 +/− 170.3

In particular illustrative aspects and embodiments of this invention, itis possible, though not necessary, to reduce the standard deviations inone or more of these PK parameters.

More uniform dissolution of progesterone in a formulation of the presentdisclosure compared to the dissolution of PROMETRIUM® at equal dosagestrengths and using the same USP apparatus can be determined usingstandard techniques established for API dissolution testing, includingthat which is described in the examples below.

Reduced intra- and inter-patient variability of progesterone formulatedpursuant to the present disclosure compared to PROMETRIUM can bedemonstrated via a fed bio-study such as that described below.

Exemplary dosage strengths for estradiol for use in the formulationsdescribed herein include, without limitation, 0.125, 0.25, 0.375, 0.50,0.625, 0.75, 1.00, 1.125, 1.25, 1.375, 1.50, 1.625, 1.75 and 2.00 mg.Exemplary dosage strengths for progesterone for use in the formulationsdescribed herein include, without limitation, 25, 50, 75, 100, 125, 150,175, 200, 250, 300, 350 and 400 mg. These dosage strengths for each ofestradiol and progesterone can be administered in formulations describedherein either alone or in combination.

In some embodiments, estradiol is solubilized. Estradiol solubilizationhelps ensure high content uniformity and enhanced stability. Solubilizedestradiol may include estradiol that is approximately 80% to 100%soluble in a solubilizing agent, including specifically embodiments thatare: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% soluble in a solubilizingagent. Solubility may be expressed as a mass fraction (% w/w, alsoreferred to as wt %).

In some embodiments, the composition comprises micronized progesterone.Progesterone active pharmaceutical ingredient may be micronized via anyone of the multiple methods typically utilized by the ordinarily skilledartisan. In various embodiments, micronized progesterone has an X50particle size value of less than about 15 microns, less than about 10microns, less than about 5 microns and/or less than about 3 microns. Invarious embodiments, micronized progesterone has an X90 particle sizevalue of less than about 25 microns, less than about 20 microns, and/orless than about 15 microns.

Particle size may be determined in any suitable manner. For example, aBeckman Coulter LS 13 320 Laser Diffraction Particle Size Analyzer (the“Beckman Device”) may be used to determine particle size. As describedabove, particle size may be represented by various metrics, for example,through an X50 particle size, and/or X90 particle size, or similardescriptions of particle size.

The Beckman Device may be used with various modules for introducing asample for analysis. The Beckman Device may be used with the LS 13 320Universal Liquid Module (“ULM”). The ULM is capable of suspendingsamples in the size range of 0.017 μm to 2000 μm. The ULM is a liquidbased module that allows for delivery of the sample to the sensing zone.The ULM recirculates the sample through the Beckman Device. The ULMcomprises two hoses, one for fluid delivery and another for waste. Thetotal volume used may be 125 mL or less. A sample mass of from about 1mg to about 10 g may be used. The ULM may interact with the BeckmanDevice via pins that fit into slots on the ULM. The ULM may use avariety of suspension fluids, for example, water, butonol, ethanol,chloroform, heptanes, toluene, propanol, COULTER Type 1B Dispersant(“Coulter 1B”), and a variety of other suspension fluids. Surfactantsmay also be used, though pump speed should be adjusted to preventexcessive bubbling. Coulter 1B may comprise one or more of acetaldehyde,ethylene oxide, and/or 1,4-dioxane. The Beckman Device may be configuredto use a variety of optical theories, including the Fraunhofer opticalmodel and the Mie Theory.

The Beckman Device may comprise software to control the Beckman Devicewhile the ULM is in use. The software may control, for example, pumpspeed, use of de-bubble routine, rinse routine, sonicate routine, andfill routine, among others. Parameters regarding the sample run may alsobe configured. For example, run length may be set. Though any suitablerun length may be used, in various embodiments, a time period of 30seconds to 120 seconds, and preferably between 30 seconds and 90 secondsmay be used.

The Beckman Device may be used with the LS 13 320 Micro Liquid Module(“MLM”). The MLM is capable of suspending samples in the size range of0.4 μm to 2000 μm. The MLM is a liquid based module that allows fordelivery of the sample to the sensing zone. The MLM includes a stirrer.The total volume used may be 12 mL or less. The MLM may use a variety ofsuspension fluids, both aqueous and non-aqueous.

In some embodiments, the progesterone is solubilized. Fully solubilizedprogesterone formulations or partially solubilized progesteroneformulations, in which at least about 50% of the progesterone, e.g.,75%, 80%, 85%, 90%, or >95% is solubilized, appear to provide improvedPK-related properties.

In some embodiments, the estradiol and the progesterone are both in thesolubilizing agent. In some embodiments, the estradiol and theprogesterone are both uniformly dispersed in the pharmaceuticalformulation.

In accordance with various aspects and embodiments, the solubilityproportion (i.e., the proportion of a solute that enters solution) isnotable. The weight ratio of estradiol to the weight of the entiresolution is also notable due to the intended dose amounts, discussedherein. In particular, it is desirable to obtain a target dosage ofestradiol in an amount of solution that may be readily administered viaa capsule. For example, if it is desired to have a dose of estradiol ina capsule of between about 0.125 mg to about 2 mg, it would also bedesirable to have a total solution weight to be between about 250 mg toabout 400 mg, preferably about 300 mg to about 350 mg and morepreferably about 325 mg. In various embodiments, the following weightratios of estradiol to total solution are from about 0.125/50 mg toabout 0.125/1000 mg, from about 1 mg:500 mg to about 1 mg:50 mg; fromabout 1 mg:250 mg to about 1 mg:60 mg; from about 1 mg:100 mg to about 1mg:66 mg; from about 2 mg/50 mg to about 2 mg/1000 mg. In variousembodiments, the target for single dose product is 325 mg, and a targetfill weight for a combination product (e.g., two or more sterol APIs) is650 mg.

In illustrative embodiments, total progesterone, i.e., dissolved andmicronized, is 20 to 50 wt %, e.g., 30 to 35 wt %; estradiol is 0.1 to0.8 wt %, e.g., 0.15 to 0.35 wt %.

Solubilizing Agents

In various embodiments, the solubilizing agent is selected from at leastone of a solvent or co-solvent. Suitable solvents and co-solventsinclude any mono-, di- or triglyceride and glycols, and combinationsthereof.

In some embodiments, formulations of the present disclosure (e.g.,estradiol and progesterone formulations) are prepared via blending witha pharmaceutically acceptable oil; generally, the oil comprises at leastone medium chain fatty acid such as medium chain fatty acids consistingof at least one mono-, di-, or triglyceride, or derivatives thereof, orcombinations thereof. Optionally added are other excipients including,for example and without limitation, anti-oxidants, lubricants and thelike. Sufficient oil is used to form a suspension of micronizedprogesterone or, in the alternative, solubilize progesterone.

In illustrative embodiments of the invention, oils used to solubilizeestradiol and to suspend, partially solubilize, or fully solubilizeprogesterone include medium chain fatty acid esters (e.g., esters ofglycerol, polyethylene glycol, or propylene glycol) and mixturesthereof. In illustrative embodiments, the medium chain fatty acids areC6 to C14 or C6 to C12 fatty acids. In illustrative embodiments, themedium chain fatty acids are saturated, or predominantly saturated,e.g., greater than about 60% or greater than about 75% saturated.

Mixtures of medium chain fatty acid glycerides, e.g., C6-C12, C8-C12, orC8-C10 fatty acid mono- and diglycerides or mono-, di-, andtriglycerides are very well suited for dissolving estradiol; goodresults have been obtained with an oil that is predominantly a mixtureof C8-C10 saturated fatty acid mono- and diglycerides. Longer chainglycerides appear to be not as well suited for dissolution of estradiol.On the other hand, high solubility of progesterone has been obtained inmixtures that are predominantly medium chain fatty acid triglycerides.

Pharmaceutically acceptable oils include, without limitation, the use ofat least one of caproic fatty acid; caprylic fatty acid; capric fattyacid; tauric acid; myristic acid; linoleic acid; succinic acid;glycerin; mono-, di-, or triglycerides and combinations and derivativesthereof; a polyethylene glycol; a polyethylene glycol glyceride(GELUCIRE®, a polyethylene glycol glyceride); GATTEFOSSE SAS,Saint-Priest, France); a propylene glycol; a caprylic/caprictriglyceride (MIGLYOL® (caprylic/capric triglyceride) SASOL GermanyGMBH, Hamburg; MIGLYOL® includes MIGLYOL® 810, 812, 816 and 829); acaproic/caprylic/capric/lauric triglyceride; a caprylic/capric/linoleictriglyceride; a caprylic/capric/succinic triglyceride; propylene glycolmonocaprylate; propylene glycol monocaprate; (CAPMUL® PG-8 (propyleneglycol monocaprylate) and 10; the CAPMUL® MCM (medium chain mono- anddiglycerides) brands are owned by ABITEC, Columbus Ohio); propyleneglycol dicaprylate; propylene glycol dicaprylate; medium chain mono- anddi-glycerides (CAPMUL® MCM); a diethylene glycol mono ester (including2-(2-Ethoxyethoxy)ethanol: TRANSCUTOL® (diethylene glycol monoethylether); esters of saturated coconut and palm kernel oil and derivativesthereof; triglycerides of fractionated vegetable fatty acids, andcombinations and derivatives thereof.

Illustrative examples of mono- and diglycerides of medium chain fattyacids include, among others, CAPMUL® MCM, CAPMUL® MCM C10, CAPMUL® MCMC8, and CAPMUL® MCM C8 EP. These oils are C8 and C10 fatty acid mono-and diglycerides. Illustrative examples of oils that are triglyceridesof medium chain fatty acids include, among others, MIGLYOL® 810 andMIGLYOL® 812.

Illustrative examples of oils that are medium chain fatty acid esters ofpropylene glycol include, among others, CAPMUL® PG-8, CAPMUL® PG-2LEP/NF, CAPMUL® PG-8 NF, CAPMUL® PG-12 EP/NF and CAPRYOL™. Otherillustrative examples include MIGLYOL® 840.

Illustrative examples of oils that are medium chain fatty acid esters ofpolyethylene glycol include, among others, GELUCIRE® 44/14 (PEG-32glyceryl laurate EP), which is polyethylene glycol glycerides composedof mono-, di- and triglycerides and mono- and diesters of polyethyleneglycol. Without intending to be bound to any particular mechanism, itappears that at least in formulations comprising small amounts ofGELUCIRE®, e.g., 10 wt % or less, the primary function of this oil is asa non-ionic surfactant.

These illustrative examples comprise predominantly medium chain length,saturated, fatty acids, specifically predominantly C8 to C12 saturatedfatty acids.

In accordance with various embodiments, the formulations of the presentdisclosure do not include peanut oil.

It will be understood that commercially available fatty acid esters ofglycerol and other glycols are often prepared from natural oils andtherefore may comprise components additional to the fatty acid estersthat comprise the predominant (by weight) component(s) and thattherefore are used to characterize the product. Such other componentsmay be, e.g., other fatty acid triglycerides, mono- and diesters, freeglycerol, or free fatty acids. So, for example, when an oil/solubilizingagent is described herein as a saturated C8 fatty acid mono- or diesterof glycerol, it will be understood that the predominant component of theoil, i.e., >50 wt % (e.g., >75 wt %, >85 wt % or >90 wt %) are caprylicmonoglycerides and caprylic diglycerides. For example, the TechnicalData Sheet by ABITEC for CAPMUL® MCM C8 describes CAPMUL® MCM C8 asbeing composed of mono and diglycerides of medium chain fatty acids(mainly caprylic) and describes the alkyl content as ≦1% C6, ≧95% C8,≦5% C10, and ≦1.5% C12 and higher.

By way of further example, MIGLYOL® 812 is generally described as aC8-C10 triglyceride because the fatty acid composition is at least about80% caprylic (C8) acid and capric (C10) acid. However, it can alsocomprise small amounts of other fatty acids, e.g., less than about 5% ofcaproic (C6) acid, lauric (C12) acid, and myristic (C14) acid.

Specifically, a product information sheet for MIGLYOL® by SASOL providesthe composition of fatty acids as follows:

TABLE 2 MIGLYOL ® Fatty Acid Composition MIGLYOL ® MIGLYOL ® MIGLYOL ®MIGLYOL ® MIGLYOL ® Fatty acid 810 812 818 829 840 Caproic acid max. 2.0max. 2.0 max. 2 max. 2 max. 2 (C6:0) Caprylic acid 65.0-80.0 50.0-65.045-65 45-55 65-80 (C8:0) Capric acid 20.0-35.0 30.0-45.0 30-45 30-4020-35 (C10:0) Lauric acid max. 2   max. 2   max. 3 max. 3 max. 2 (C12:0)Myristic acid max. 1.0 max. 1.0 max. 1 max. 1 max. 1 (C14:0) Linoleicacid — — 2-5 — — (C18:2) Succinic acid — — — 15-20 —

Where certain embodiments of this invention are described as comprising(or consisting essentially of) a capsule shell, estradiol solubilized inC8-C10 triglycerides, and a thickening agent, it will be understood thatthe fatty acid esters component of the formulation may be, e.g.,MIGLYOL® 812 or a similar product.

By way of further illustration, GELUCIRE® 44/14 is generally describedas lauroyl polyoxyl-32 glycerides, i.e., polyoxyethylene 32 lauricglycerides (which is a mixture of mono-, di-, and triesters of glyceroland mono- and diesters of PEGs) because the fatty acid composition is 30to 50% lauric acid and smaller amounts of other fatty acids, e.g., up to15% caprylic acid, up to 12% capric acid, up to 25% myristic acid, up to25% palmitic acid, and up to 35% stearic acid. The product may alsocontain small amounts of non-esterified glycols. Where certainembodiments of this invention are described as described as comprising(or consisting essentially of) a capsule shell, estradiol solubilized intriglycerides, and a thickening agent that is a non-ionic surfactantcomprising C8 to C18 fatty acid esters of glycerol and polyethyleneglycol, it will be understood that the thickening agent component of theformulation may be, e.g., GELUCIRE® 44/14 or a similar product.

Similarly, where certain embodiments of this invention are as describedas comprising (or consisting essentially of) a capsule shell, estradiolsolubilized in triglycerides, and a thickening agent that is a non-ionicsurfactant comprising PEG-6 stearate, ethylene glycol palmitostearate,and PEG-32 stearate, it will be understood that the thickening agentcomponent of the formulation may be, e.g., TEFOSE® 63 or a similarproduct.

In addition to the oils referenced above, other solubilizers include,for example and without limitation, glyceryl mono- and di-caprylates,propylene glycol and 1,2,3-propanetriol (glycerol, glycerin, glycerine).

Anionic and/or non-ionic surfactants can be used in other embodiments ofthe presently disclosed formulations containing estradiol, progesteroneor a combination thereof. In certain embodiments, a non-ionic surfactantis used. Exemplary non-ionic surfactants may include, for example andwithout limitation, one or more of oleic acid, linoleic acid, palmiticacid, and stearic acid esters or alcohols. In further embodiments, thenon-ionic surfactant may comprise polyethylene sorbitol esters,including polysorbate 80, which is commercially available under thetrademark TWEEN 80® (Sigma Aldrich, St. Louis, Mo.). Polysorbate 80comprises approximately 60%-70% oleic acid with the remainder comprisingprimarily linoleic acids, palmitic acids, and stearic acids. Polysorbate80 may be used in amounts ranging from about 5 to 50%, and in certainembodiments, about 30% of the formulation total mass.

In various other embodiments, the non-ionic surfactant is selected fromone or more of glycerol and polyethylene glycol esters of fatty acids,for example, lauroyl macrogol-32 glycerides and/or lauroyl polyoxyl-32glycerides, commercially available as GELUCIRE®, including, for example,GELUCIRE® 44/11 and GELUCIRE® 44/14. These surfactants may be used atconcentrations greater than about 0.01%, and typically in variousamounts of about 0.01%-10.0%, 10.1%-20%, and 20.1%-30%. In certainexamples, below, GELUCIRE® 44/14 is used as a surfactant in amounts of 1to 10 wt %. See, e.g., Tables 13-17, below. Other non-ionic surfactantsinclude, e.g., LABRASOL® PEG-8 Caprylic/Capric Glycerides (Gattefosse)and LABARAFIL® corn/apricot oil PEG-6 esters (Gattefosse).

Other Excipients

In other embodiments, a lubricant is used. Any suitable lubricant may beused, such as for example lecithin. Lecithin may comprise a mixture ofphospholipids.

In additional embodiments, an antioxidant is used. Any suitableantioxidant may be used such as, for example and without limitation,butylated hydroxytoluene.

For example, in various embodiments, a pharmaceutical formulationcomprises about 20% to about 80% carrier by weight, about 0.1% to about5% lubricant by weight, and about 0.01% to about 0.1% antioxidant byweight.

The choice of excipient will, to a large extent, depend on factors suchas the particular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form. Excipientsused in various embodiments may include colorants, flavoring agents,preservatives and taste-masking agents. Colorants, for example, maycomprise about 0.1% to about 2% by weight. Preservatives may comprisemethyl and propyl paraben, for example, in a ratio of about 10:1, and ata proportion of about 0.005% and 0.05% by weight.

As is with all oils, solubilizers, excipients and any other additivesused in the formulations described herein, each is to be non-toxic andpharmaceutically acceptable.

Formulation and Administration

In some embodiments, combinations of solubilizing agents (e.g., two ormore oils or combinations of one or more oils and one or moresurfactants) are used to form estradiol and progesterone compositions.Various ratios of these solubilizing agents (e.g., oils or surfactants)can be used. CAPMUL® MCM and a non-ionic surfactant, e.g., GELUCIRE®44/14 (lauroyl macrogol-32 glycerides EP; lauroyl polyoxyl-32 glyceridesNF; lauroyl polyoxylglycerides (USA FDA IIG)), can be used at ratios ofabout 99:1 to 2:1, including, for example and without limitation: 60:40,65:35, 70:30, 75:25, 80:10, 80:15, 85:20, 90:10, and 98:1. The ratios ofoil (e.g., medium chain fatty acid esters of monoglycerides anddiglycerides) to non-ionic surfactant can be significantly higher. Forexample, in certain examples, below, CAPMUL® MCM and GELUCIRE® were usedin ratios of up to about 65:1, e.g., 8:1, 22:1, 49:1, 65:1 and 66:1.See, e.g., Tables 20-24, below. Thus, useful ratios can be 8:1 orgreater, e.g., 60 to 70:1. Among other combinations, these solubilizers,as defined herein, and combinations thereof, can be used to formcombination estradiol and progesterone formulations of the presentdisclosure.

In illustrative embodiments, estradiol or progesterone is soluble in theoils at room temperature, although it may be desirable to warm the oilsup until they are in a liquid state. In illustrative embodiments, theoil or oil/surfactant is liquid at between room temperature and about50° C., e.g., at or below 50° C., at or below 40° C., or at or below 50°C. In illustrative embodiments, GELUCIRE® 44/14 is heated to about 65°C. and CAPMUL® MCM is heated to about 40° C. to facilitate mixing of theoil and non-surfactant, although such heating is not necessary todissolve the estradiol or progesterone. In illustrative embodiments, thesolubility of estradiol in the oil (or oil/surfactant) is at least about0.5 wt %, e.g., 0.8 wt % or higher, or 1.0 wt % or higher. However, muchhigher solubility can be achieved. For example, as shown in Example 4,below, estradiol is stable in solution in CAPMUL® MCM at 12 mg/g (whichis approximately equal to 12 mg/ml). As shown in Example 19, suchsolubility is favored over results observed in longer chain andunsaturated fatty acid esters.

High solubility of estradiol has been obtained in2-(2-Ethoxyethoxy)ethanol, e.g., TRANSCUTOL® and in Propylene glycolmonocaprylate, e.g., CAPRYOL™ 90 (Gattefosse).

In some embodiments, progesterone is fully solubilized using, forexample and without limitation, sufficient amounts of: TRANSCUTOL® andMIGLYOL®; TRANSCUTOL®, MIGLYOL® and CAPMUL® PG 8 and/or PG 10; CAPMUL®MCM; CAPMUL® and a non-ionic surfactant; and CAPMUL® MCM and GELUCIRE®.

Combinations of these oils can produce partially solubilizedprogesterone, depending upon the desired unit dosage amount ofprogesterone. The greater the amount of progesterone per unit dosageform, the less progesterone may be solubilized. The upward limit ofdosage strength per unit dose it generally limited only by the practicalsize of the final dosage form.

In illustrative embodiments of the invention, the selected oil does notrequire excessive heating in order to solubilize progesterone orestradiol. For example, when the formulation comprises medium chainfatty acid mono- and diglycerides (e.g., CAPMUL® MCM) and polyethyleneglycol glycerides (e.g., GELUCIRE®) as a surfactant, the oil and/or thesurfactant can be warmed up, e.g., to about 65° C. in the case of thesurfactant and less in the case of the oil, to facilitate mixing of theoil and surfactant. The estradiol can be added at this temperature or atlower temperatures as the mixture cools or even after it has cooled astemperatures above room temperature, e.g., about 20° C., are notrequired to solubilize the estradiol in preferred oils. The progesteronecan also be added as the mixture cools, e.g., to below about 40° C. orto below about 30° C., even down to room temperature.

As a non-limiting example, an illustrative embodiment of apharmaceutical composition of the invention comprises solubilizedestradiol, progesterone at least 75% of the progesterone beingsolubilized (the balance being micronized as discussed elsewhereherein), and an oil, wherein the oil is medium chain fatty acid mono-and diesters of glycerol, with or without surfactant. In certainembodiments, a specification for progesterone is set at >80%solubilized, <20% micronized or >85% solubilized, <15% micronized.Specific examples of such illustrative embodiments, with GELUCIRE® assurfactant, in which at least about 85% of the progesterone can besolubilized, include, e.g., the following four formulations:

TABLE 3 Formulation A—P:50/E2:0.25: Amount Qty/Capsule Ingredient(s) (%w/w) (mg) Progesterone, USP, 33.33 50.00 micronized EstradiolHemihydrate 0.17 0.26 CAPMUL ® MCM, NF 65.49 98.24 GELUCIRE ® 44/14, NF1.00 1.50 Total 100.00 150.00

TABLE 4 Formulation B—P:50/E2:0.5: Amount Qty/Capsule Ingredient(s) (%w/w) (mg) Progesterone, USP, 33.33 50.00 micronized EstradiolHemihydrate 0.35 0.52 CAPMUL ® MCM, NF 65.32 97.98 GELUCIRE ® 44/14, NF1.00 1.50 Total 100.00 150.00

TABLE 5 Formulation C—P:100/E2:0.5: Amount Qty/Capsule Ingredient(s) (%w/w) (mg) Progesterone, USP, 33.33 100.00 micronized EstradiolHemihydrate 0.17 0.52 CAPMUL ® MCM, NF 65.49 196.48 GELUCIRE ® 44/14, NF1.00 3.00 Total 100.00 300.00

TABLE 6 Formulation D—P:100/E2:1: Amount Qty/Capsule Ingredient(s) (%w/w) (mg) Progesterone, USP, 33.33 100.00 micronized EstradiolHemihydrate 0.34 1.03 CAPMUL ® MCM, NF 65.32 195.97 GELUCIRE ® 44/14, NF1.00 3.00 Total 100.00 300.00

TABLE 7 Formulation E—P:200/E2:2: Amount Qty/Capsule Ingredient(s) (%w/w) (mg) Progesterone, USP, 33.33 200.00 micronized EstradiolHemihydrate 0.34 2.06 CAPMUL ® MCM, NF 65.32 391.94 GELUCIRE ® 44/14, NF1.00 6.00 Total 100.00 600.00Note: 1.00 mg Estradiol equivalent to 1.03 mg Estradiol Hemihydrate.

In general terms, the above formulations comprise 30 to 35 wt %progesterone, 0.1 to 0.4 wt % estradiol (or estradiol hemihydrate), 55to 75 wt % of an oil that is predominantly medium chain fatty acid mono-and diglycerides, such as CAPMUL® MCM, and 0.5 to 10 wt % non-ionicsurfactant, such as GELUCIRE® 44/14. The above formulations may bemodified to comprise excipients, e.g., gelatin such as Gelatin 200Bloom, glycerin, coloring agents such as Opatint red and white, and,optionally, MIGLYOL® 812.

Estradiol solubilization helps ensure high content uniformity andenhanced stability. Fully solubilized progesterone formulations orpartially solubilized progesterone formulations in which at least about50% of the progesterone, e.g., 75%, 80%, 85%, 90%, or >95%, issolubilized appear to provide improved PK-related properties.

Pharmaceutical formulations as described herein can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidpreparation can comprise one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Mack Publishing Co, Easton Pa.(“Remington's”).

In some embodiments, the pharmaceutical formulations described hereinare prepared and administered as filled capsules, typically softcapsules of one or more materials well known in the art including, forexample and without limitation, soft gelatin capsules. Micronizedprogesterone, as described herein, may also be prepared foradministration in tablets or other well-known orally administered dosageforms using standard techniques.

In some embodiments, in which the carrier is a medium fatty acid esterof a glycol and which comprise a non-ionic surfactant as describedherein, the formulations are in liquid form, i.e., not gels, hard fatsor other solid forms.

In general, the type of composition is selected based on the mode ofadministration. A pharmaceutical composition (e.g., for oraladministration or delivery by injection) can be in the form of a liquid(e.g., an elixir, syrup, solution, emulsion or suspension).Alternatively, a pharmaceutical composition as described herein can takethe form of a pill, tablet, or capsule containing the liquid oil, andthus, the composition can contain any of the following: a diluent suchas lactose, sucrose, dicalcium phosphate, and the like; a disintegrantsuch as starch or derivatives thereof; a lubricant such as magnesiumstearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. Thecomposition can also be formulated into a suppository disposed, forexample, in a polyethylene glycol (PEG) solubilizing agent.

Administration of the formulations described herein can be carried outvia any of the accepted modes of administration. Thus, administrationcan be, for example, intravenous, topical, subcutaneous, transcutaneous,transdermal, intramuscular, oral, intra-joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, or byinhalation. In some embodiments, a composition as described herein isadministered orally. For example, a pharmaceutical composition asdescribed herein can be administered via capsules such as soft capsules.In some embodiments, a pharmaceutical composition as described herein isadministered once daily.

According to various embodiments described herein, a 28-day or monthlyregimen of capsules can be packaged in a single kit (e.g., a blisterpack) having administration days identified to improve compliance andreduce associated symptoms, among others. One or more of the capsulesmay contain no estradiol, for example, and/or no progesterone. Capsulesthat comprise no estrogen or progesterone API may be referred to asplacebos. A blister pack can have a plurality of scores or perforationsseparating blister pack into 28 days. Each day may further comprise asingle blister or a plurality of blisters. In various embodiments, eachunit dose may contain micronized and/or partially solubilized, or fullysolubilized progesterone and/or solubilized estradiol in amounts as setforth herein above, although other dose ranges may be contemplated. Inaddition, kits having other configurations are also contemplated herein.For example, without limitation, kits having such blister packs maycontain any number of capsules.

As referenced above, the formulations of the present disclosure aregenerally orally administered, typically via, for example, capsules suchas soft capsules. The present formulations can also be used to formtransdermal patches using standard technology known in the art.Solubilized formulations of the present invention can also be formulatedfor intraperitoneal administration using techniques well known in theart.

In some embodiments, the pharmaceutical formulations disclosed hereinare useful in treating conditions in subjects caused, at least in part,by estrogen deficiency, particularly for women with a uterus. Forexample, in some embodiments, the formulations disclosed herein areuseful for the treatment of an animal, especially a mammal, includinghumans, for menopause; for endometrial hyperplasia; for secondaryamenorrhea; as a method of treatment for preterm birth, when said animalhas a shortened cervix, and other disease states or conditions treatedwith supplemental progesterone or estrogen. In some embodiments, aformulation as disclosed herein is useful for treating one or moresymptoms of menopause such as vaginal atrophy, vaginal dryness, waterydischarge, skin dryness, osteoporosis, thin bones, painful fractures,incontinence, urinary frequency, urinary urgency, urinary tractinfections, temperature dyregulation (e.g., feeling unusually hot orcold or temperature swings), dysfunctional bleeding, rapid heartbeat,migraine, breast tenderness or swelling, breast atrophy, decreased skinelasticity, back pain, joint pain, muscle pain, fatigue, decreasedlibido, dyspareunia, vasomotor symptoms (e.g., flushing, hot flashes),irritability, memory loss, mood disturbance, depression, anxiety, sleepdisturbance, and sweating. Thus, in some embodiments, the presentdisclosure provides methods of treating such a condition byadministering to the subject a composition comprising estradiol andprogesterone as described herein. As used herein, the term “treatment,”or a derivative thereof, contemplates partial or complete inhibition ofthe stated disease state when a formulation as described herein isadministered prophylactically or following the onset of the diseasestate for which such formulation is administered. For the purposes ofthe present disclosure, “prophylaxis” refers to administration of theactive ingredient(s) to an animal, especially a mammal, to protect theanimal from any of the disorders set forth herein, as well as others.

Bioavailability Properties

The pharmaceutical formulations of the present disclosure can beformulated to provide desirable pharmacokinetic parameters in a subject(e.g., a female subject) to whom the composition is administered. Insome embodiments, a pharmaceutical composition as described hereinproduces desirable pharmacokinetic parameters for progesterone in thesubject. In some embodiments, a pharmaceutical composition as describedherein produces desirable pharmacokinetic parameters for estradiol inthe subject. In some embodiments, a pharmaceutical composition asdescribed herein produces desirable pharmacokinetic parameters for oneor more metabolites of progesterone or estradiol in the subject, forexample estrone or total estrone.

In certain embodiments, combination formulations of the presentdisclosure exhibit bioavailability properties that are comparable to thebioavailability properties of the components of the combinationformulation when individually formulated (e.g., progesterone in a peanutoil, e.g., PROMETRIUM®, and a micronized estradiol tablet, e.g.,ESTRACE®). In certain embodiments, a composition is within the scope ofthe invention if it has a value for a pharmacokinetic parameter that isabout 80% to about 125% of the value of the pharmacokinetic parameterfor a reference composition when said formulation is administered to ahuman subject.

Following the administration of a composition comprising progesteroneand estradiol to a subject, the concentration and metabolism ofprogesterone or estradiol can be measured in a sample (e.g., a plasmasample) from the subject. Progesterone is metabolized to pregnanediolsand pregnanolones, which are then conjugated to glucuronide and sulfatemetabolites that are excreted or further recycled. Estradiol isconverted reversibly to estrone, and both estradiol and estrone can beconverted to the metabolite estriol. In postmenopausal women, asignificant proportion of circulating estrogens exist as sulfateconjugates, especially estrone sulfate. Thus, estrone can be measuredwith respect to “estrone” amounts (excluding conjugates such as estronesulfate) and “total estrone” amounts (including both free, orunconjugated, estrone and conjugated estrone such as estrone sulfate).

The pharmaceutical formulations of the present disclosure can becharacterized for one or more pharmacokinetic parameters ofprogesterone, estradiol, or a metabolite thereof followingadministration of the composition to a subject or to a population ofsubjects. These pharmacokinetic parameters include AUC, C_(max), andT_(max).

In certain embodiments, a composition is within the scope of theinvention if it has a C_(max) value that is about 80% to about 125% ofthe C_(max) value of the reference composition. C_(max) is wellunderstood in the art as an abbreviation for the maximum drugconcentration in serum or plasma of the test subject. In certainembodiments, a composition is within the scope of the invention if ithas a T_(max) value that is about 80% to about 125% of the T_(max) valueof the reference composition. T_(max) is well understood in the art asan abbreviation for the time to maximum drug concentration in serum orplasma of the test subject. In vivo testing protocols for determining aC_(max) and/or T_(max) value can be designed in a number of ways.

In certain embodiments, a composition is within the scope of theinvention if it has an AUC value that is about 80% to about 125% of theAUC value of the reference composition. AUC is a determination of thearea under the curve (AUC) plotting the serum or plasma concentration ofdrug along the ordinate (Y-axis) against time along the abscissa(X-axis). AUCs are well understood, frequently used tools in thepharmaceutical arts and have been extensively described, for example in“Pharmacokinetics Processes and Mathematics,” Peter E. Welling, ACSMonograph 185; 1986.

Any of a variety of methods can be used for measuring the levels ofprogesterone, estradiol, estrone, or total estrone in a sample,including immunoassays, mass spectrometry (MS), high performance liquidchromatography (HPLC) with ultraviolet fluorescent detection, liquidchromatography in conjunction with mass spectrometry (LC-MS), tandemmass spectrometry (MS/MS), and liquid chromatography-tandem massspectrometry (LC-MS/MS). It will be understood by a person of skill inthe art that the sensitivity of the assay used will correlate with thelevel of quantification that can be detected, and that a moresensitivity assay will enable lower levels of quantification ofprogesterone, estradiol, estrone, or total estrone. In some embodiments,the levels of progesterone, estradiol, estrone, or total estrone aremeasured using a validated LC-MS/MS method. Methods of measuring hormonelevels are well described in the literature.

The levels of progesterone, estradiol, estrone, or total estrone can bemeasured in any biological sample, e.g. a tissue or fluid such as blood,serum, plasma, or urine. In some embodiments, the sample is blood orplasma. In some embodiments, the levels of progesterone, estradiol,estrone, or total estrone are measured about 0.0, 0.10, 0.20, 0.05,0.30, 0.35, 0.40, 0.45, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18,21, 24, 27, 30, 33, 36, 39, 42, 45, or 48 hours after dosing, or anyother appropriate time period that is common or useful in determiningthe levels of each of the hormones. Generally, assays to determine thelevels of progesterone, estradiol, estrone, or total estrone aremeasured one or more times every 5, 10, 15, 20, 30, 60, 120, 360, 480,720, or 1440 minutes after administration, or combinations thereof(e.g., the first measurements are taken every 15 minutes for the firsthour, followed by every 120 minutes thereafter). In some embodiments,the levels of progesterone, estradiol, estrone, or total estrone aremeasured about 48 hours after dosing. In embodiments, the timing of suchmeasurements are designed to accurately measure C_(max), T_(max), orAUC. Timing can be adjusted based on the given circumstances (i.e., oneformulation may cause a more rapid C_(max), in which case the initialtimes would be clustered closer together, closer to time zero, or bothto ensure accurate measurement of C_(max), T_(max), and AUC).

In some embodiments, the values for C_(max), T_(max), and/or AUCrepresent a number of values taken from all the subjects in a patienttest population and are, therefore, mean values averaged over the entiretest population. Alternatively, the C_(max) value, T_(max) value, and/orAUC test/AUC control ratio may be determined for each subject, thenaveraged.

In some embodiments, administration of the pharmaceutical formulation asdisclosed herein (e.g., a pharmaceutical formulation comprisingsolubilized estradiol, suspended progesterone, and a medium chain(C6-C12) oil) to a subject produces, in a plasma sample from thesubject, one or more parameters selected from:

(i) an AUC_((0-t)) for estradiol that is from 1123 pg·h/ml to 1755pg·h/ml;(ii) an AUC_((0-∞)) for estradiol that is from 1968 pg·hr/ml to 3075pg·hr/ml; or(iii) a C_(max) for estradiol that is from 52 pg/ml to 81 pg/ml.

In some embodiments, administration of the formulation to the subjectproduces both an AUC_((0-t)) for estradiol that is from 1123 pg·h/ml to1755 pg·h/ml, and a C_(max) for estradiol that is from 52 pg/ml to 81pg/ml.

In some embodiments, administration of the formulation to the subjectfurther produces, in a plasma sample from the subject, one or moreparameters selected from:

(i) an AUC_((0-t)) for progesterone that is from 96 ng·hr/ml to 150ng·hr/ml;(ii) an AUC_((0-∞)) for progesterone that is from 105 ng·hr/ml to 164ng·hr/ml; or(ii) a C_(max) for progesterone that is from 71 ng/ml to 112 ng/ml.

In some embodiments, administration of the composition to the subjectproduces both an AUC_((0-t)) for progesterone that is from 96 ng·hr/mlto 150 ng·hr/ml, and a C_(max) for progesterone that is from 71 ng/ml to112 ng/ml.

In some embodiments, administration of the formulation to the subjectproduces, in a plasma sample from the subject,

(i) an AUC_((0-t)) for estradiol that is from 1123 pg·h/ml to 1755pg·h/ml;(ii) a C_(max) for estradiol that is from 52 pg/ml to 81 pg/ml;(iii) an AUC_((0-t)) for progesterone that is from 96 ng·hr/ml to 150ng·hr/ml; or(iv) a C_(max) for progesterone that is from 71 ng/ml to 112 ng/ml.

In some embodiments, administration of the pharmaceutical formulation tothe subject produces, in a plasma sample from the subject, one, two,three or more parameters selected from:

(i) an AUC_((0-t)) for estradiol that is from 1123 pg·h/ml to 1755pg·h/ml;(ii) a C_(max) for estradiol that is from 52 pg/ml to 81 pg/ml;(iii) an AUC_((0-t)) for progesterone that is from 96 ng·hr/ml to 150ng·hr/ml; or(iv) a C_(max) for progesterone that is from 71 ng/ml to 112 ng/ml.

In some embodiments, administration of the pharmaceutical formulation tothe subject produces both parameters (i) and (ii). In some embodiments,administration of the formulation to the subject produces bothparameters (i) and (iii). In some embodiments, administration of theformulation to the subject produces both parameters (i) and (iv). Insome embodiments, administration of the formulation to the subjectproduces both parameters (ii) and (iii). In some embodiments,administration of the formulation to the subject produces bothparameters (ii) and (iv). In some embodiments, administration of theformulation to the subject produces both parameters (iii) and (iv). Insome embodiments, administration of the formulation to the subjectproduces all of parameters (i), (ii), and (iii). In some embodiments,administration of the formulation to the subject produces bothparameters (i), (iii), and (iv). In some embodiments, administration ofthe formulation to the subject produces both parameters (ii), (iii), and(iv). In some embodiments, administration of the formulation to thesubject produces all of parameters (i), (ii), (iii), and (iv).

In some embodiments, administration of the pharmaceutical formulation tothe subject further produces, in a plasma sample from the subject, oneor more parameters selected from:

(i) an AUC_((0-t)) for estrone sulfate that is from 7277 pg·hr/ml to11370 pg·hr/ml;(ii) an AUC_((0-∞)) for estrone sulfate that is from 9596 pg·hr/ml to14994 pg·hr/ml; or(ii) a C_(max) for estrone sulfate that is from 341 pg/ml to 533 pg/ml.

In some embodiments, administration of the pharmaceutical composition tothe subject further produces, in a plasma sample from the subject, oneor more parameters selected from:

(i) an AUC_((0-t)) for total estrone that is from 161 pg·h/ml to 252pg·h/ml;(ii) an AUC_((0-∞)) for total estrone that is from 171 pg·hr/ml to 267pg·hr/ml; or(ii) a C_(max) for total estrone that is from 28 pg/ml to 44 pg/ml.

In some embodiments, the pharmaceutical composition is administered to apopulation of subjects in need thereof, and mean parameters aredetermined for samples (e.g., plasma samples) from the subjectsadministered the composition. Thus, in some embodiments, administrationof the composition to a population of subject produces, in plasmasamples from the subjects, one or more of a mean AUC_((0-t)) forestradiol that is from 1123 pg·h/ml to 1755 pg·h/ml, a mean AUC_((0-∞))for estradiol that is from 1968 pg·hr/ml to 3075 pg·hr/ml, or a meanC_(max) for estradiol that is from 52 pg/ml to 81 pg/ml. In someembodiments, administration of the composition to a population ofsubject produces, in plasma samples from the subjects, one or more of amean AUC_((0-t)) for progesterone that is from 96 ng·hr/ml to 150ng·hr/ml, a mean AUC_((0-∞)) for progesterone that is from 105 ng·hr/mlto 164 ng·hr/ml, or a mean C_(max) for progesterone that is from 71ng/ml to 112 ng/ml. In some embodiments, administration of thecomposition to a population of subject produces, in plasma samples fromthe subjects, one or more of a mean AUC_((0-t)) for estrone sulfate thatis from 7277 pg·hr/ml to 11370 pg·hr/ml, a mean AUC_((0-∞)) for estronesulfate that is from 9596 pg·hr/ml to 14994 pg·hr/ml, or a mean C_(max)for estrone sulfate that is from 341 pg/ml to 533 pg/ml. In someembodiments, administration of the composition to a population ofsubject produces, in plasma samples from the subjects, one or more of amean AUC_((0-t)) for total estrone that is from 161 pg·h/ml to 252pg·h/ml, a mean AUC_((0-t)) for total estrone that is from 171 pg·hr/mlto 267 pg·hr/ml, or a mean C_(max) for total estrone that is from 28pg/ml to 44 pg/ml.

In some embodiments, method of treating a subject are provided. In someembodiments, the method comprises administering to the subject apharmaceutical composition as described herein, wherein administrationof the pharmaceutical composition produces, in a plasma sample from thesubject, one or more parameters selected from: an AUC_((0-t)) forestradiol that is from 1123 pg·h/ml to 1755 pg·h/ml; an AUC_((0-∞)) forestradiol that is from 1968 pg·hr/ml to 3075 pg·hr/ml; a C_(max) forestradiol that is from 52 pg/ml to 81 pg/ml; an AUC_((0-t)) forprogesterone that is from 96 ng·hr/ml to 150 ng·hr/ml; an AUC_((0-∞))for progesterone that is from 105 ng·hr/ml to 164 ng·hr/ml; a C_(max)for progesterone that is from 71 ng/ml to 112 ng/ml; an AUC_((0-t)) forestrone sulfate that is from 7277 pg·hr/ml to 11370 pg·hr/ml; anAUC_((0-∞)) for estrone sulfate that is from 9596 pg·hr/ml to 14994pg·hr/ml; a C_(max) for estrone sulfate that is from 341 pg/ml to 533pg/ml; an AUC_((0-t)) for total estrone that is from 161 pg·h/ml to 252pg·h/ml; an AUC_((0-∞)) for total estrone that is from 171 pg·hr/ml to267 pg·hr/ml; and a C_(max) for total estrone that is from 28 pg/ml to44 pg/ml.

III. EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed subject matter.

Example 1 Estradiol Solubility

In various experiments, suitable solvents were determined for providingsufficient solubility to make 2 mg of estradiol in a 100 mg fill mass,with a desired goal of achieving ˜20 mg/g solubility for estradiol.Initial solubility experiments were done by mixing estradiol withvarious solvents, saturate the solution with the estradiol, equilibratefor at least 3 days and filter the un-dissolved particles and analyzingthe clear supernatant for the amount of estradiol dissolved by HPLC.

Estradiol solubility experiments were performed. From this list at leastone item (e.g. propylene glycol) is known to be unsuitable forencapsulation in more than 20% w/w concentration.

TABLE 8 Ingredient Solubility (mg/g) PEG 400 105*  Propylene Glycol 75*Polysorbate 80 36* TRANSCUTOL ® HP 141 CAPMUL ® PG8 31.2 *Literaturereference—Salole, E.G. (1987) The Physicochemical Properties ofOestradiol, J Pharm and Biomed Analysis, 5, 635-640.

In further solubility studies, estradiol was soluble at least 6 mg/gmMIGLYOL® TRANSCUTOL® in ratios of 81:19 to 95:5, in MIGLYOL®; ethanol at91:11, and in MIGLYOL®:CAPMUL® PG8 at 88:11, but not inMIGLYOL:TRANSCUTOL at 96:4, MIGLYOL®:LABRASOL® at 70:30 to 80:20, orMIGLYOL®:CAPMUL® PG8 at 86:14.

Example 2

It was desired to achieve 50 mg of progesterone suspended in a mediumthat can also solubilize 2 mg estradiol in a total capsule fill mass of200 mg. In order to achieve this formulation, the required solubility ofestradiol needs to be ˜10 mg/g. A total fill weight of 200 mg wasconsidered suitable for a size 5 oval soft gelatin capsule.

Additional solubility studies were performed to find solvent mixturesthat might possibly be more suitable for soft gelatin encapsulation.Solubility studies were conducted with CAPMUL® PG8 and CAPMUL® MCM bymixing estradiol with various solvent systems and as before by analyzingfor the amount of estradiol dissolved by HPLC after filtration. Resultsof these experiments are presented in Table 9. It can be seen from theseresults that mixtures containing MIGLYOL®:CAPMUL® PG8 at 50%; and alsoCAPMUL® MCM alone or in combination with 20% Polysorbate 80 can achievesufficient solubility to meet the target of 10 mg/g. CAPMUL® PG8 mixedwith MIGLYOL® at the 15 and 30% level did not provide sufficientsolubility.

TABLE 9 Ingredient Solubility (mg/g) MIGLYOL ®:CAPMUL ® PG8 (85:15) 4.40MIGLYOL ®:CAPMUL ® PG8 (70:30) 8.60 TRANSCUTOL:MIGLYOL ® 812: >12 CAPMULPG8 (5:65:28) TRANSCUTOL ®:MIGLYOL ® 812: >12 CAPMUL ® PG8 (5:47:47)MIGLYOL ®:CAPMUL ® PG8 (50:50) 14.0 CAPMUL ® MCM 19.8 Polysorbate80:CAPMUL ® MCM (20:80) 15.0

Example 3

Additional studies were performed to assess the stability of estradiol(4-6 mg) in solvent mixtures, as reported in Table 10. MIGLYOL® 812 with4% TRANSCUTOL®precipitated on Hot/Cold cycling after 96 hours, whileestradiol solubilized in MIGLYOL®:CAPMUL® blends at 30 and 50% or inCAPMUL® MCM alone, did not precipitate under the same conditions for aminimum of 14 days.

TABLE 10 Estradiol Results Hot/Cold Formulation (mg/g) CyclingTRANSCUTOL ®: 4 Crystallizes after MIGLYOL ® 812 (4:96) 96 hoursMIGLYOL ® 812: 6 Clear, after 14 days CAPMUL ® PG8 (70:30) MIGLYOL 812:6 Clear, after 14 days CAPMUL ® PG8 (50:50) TRANSCUTOL ®:MIGLYOL ® 6Clear, after 14 days 812:CAPMUL ® PG8 (5:80:15) CAPMUL ® MCM 6 Clear,after 14 days

As shown in Table 11 below, it was found that 12 mg estradiolsolubilized in MIGLYOL®:CAPMUL® PG8 50:50, CAPMUL® MCM, and in mixturesof TRANSCUTOL®: MIGLYOL®: CAPMUL® PG8 are stable and do not precipitatefor at least 12 days.

TABLE 11 Estradiol Results Hot/Cold Formulation (mg/g) Cycling MIGLYOL ®812: 12 Clear, after 12 days CAPMUL ® PG8 (50:50) TRANSCUTOL ®: 12Clear, after 12 days MIGLYOL ® 812: CAPMUL ® PG8 (5:65:28) TRANSCUTOL ®:12 Clear, after 12 days MIGLYOL ® 812: CAPMUL ® PG8 (5:47:47) CAPMUL ®MCM 12 Clear, after 12 days

Example 4

In addition to determining physical stability of the estradiol solutionsover time, it is necessary to determine if the fill material will bestable during the encapsulation process. One way to test thesepreparations is with the addition of water to the fill mass. As can beseen in Table 12, estradiol solutions at a concentration of 6 mg/g inPolyethylene Glycol 400 and CAPMUL® MCM are able to absorb a minimum of7% water without recrystallization, whereas the same concentration inMIGLYOL® 812:CAPMUL® PG8 (75:25) precipitates.

Estradiol solutions at a concentration of 12 mg/g in Polyethylene Glycol400 and CAPMUL® MCM are able to absorb a minimum of 7% water withoutrecrystallization. All CAPMUL® PG8 containing formulations turned hazyon the addition of water. However, it should be noted that estradiolrecrystallization was not observed, and the addition of water to CAPMUL®PG 8 alone (without any estradiol) also turns hazy on the addition ofwater.

TABLE 12 Estradiol Results after addition Formulation (mg/g) of 7% waterMIGLYOL ® 812:CAPMUL ® 6 Precipitated PG8 (75:25) MIGLYOL ® 812:CAPMUL ®12 Hazy PG8 (50:50) TRANSCUTOL ®:MIGLYOL ® 12 Hazy 812:CAPMUL ® PG8(5:65:28) CAPMUL ® MCM 12 Clear TRANSCUTOL ®:MIGLYOL ® 12 Hazy812:CAPMUL ® PG8 (5:47:47) Polyethylene Glycol 400 12 clear

Example 5

In an exemplary embodiment, a capsule is provided containing a fillmaterial comprising:

TABLE 13 Ingredient Mg/Capsule Estradiol Hemihydrate 2.00 Triglycerideof caprylic/capric acid qs (e.g., MIGLYOL ® 812) Diethylene GlycolMonoethylether 65.00 (TRANSCUTOL ® HP) Liquid lecithin 1.63 ButylatedHydroxytoluene 0.13 Total Fill Weight 325

Example 6

In an exemplary embodiment, a capsule is provided containing a fillmaterial comprising:

TABLE 14 Ingredient Mg/Capsule Estradiol Hemihydrate 2.00Monoglycerides/diglycerides of capric acid qs (e.g., CAPMUL ® MCM)Liquid lecithin 1.63 Polysorbate 80 97.5 Total Fill Weight 325

In an exemplary embodiment, a capsule is provided containing a fillmaterial comprising:

TABLE 15 Ingredient Mg/Capsule % w/w Amount/Batch Estradiol Hemihydrate2.03 0.62 20.2 g  Monoglycerides/diglycerides 322.97 99.38 3.23 kg ofcapric acid (e.g., CAPMUL ® MCM) Total 100 3.25 kg

The above formulation is prepared as follows: estradiol is added toCAPMUL® MCM and mixed until dissolved.

Example 7 Progesterone Solubility

In various embodiments, both estradiol and progesterone may be dissolvedin a solvent. In various embodiments, the solubility of both estradioland progesterone will be such that a therapeutically effective dose maybe obtained in a reasonably sized mass, generally considered to bebetween 1 mg and 1200 mg, preferably suitable for encapsulation in asize 3 to 22 oval or oblong capsule. For example, in variousembodiments, 50 mg to 100 mg of progesterone may be dissolved in avolume of solvent; i.e., the solubility would be 50 mg to 100 mg percapsule. MIGLYOL® was attempted, and while it can be considered a goodcarrier for progesterone, it alone did not provide a desirable level ofsolubilization of estradiol (e.g., solubility of 12 mg/g may bedesirable in various embodiments). Thus, MIGLYOL® may be used inembodiments comprising a suspension of progesterone, though MIGLYOL®,standing alone, is not desirable for use in embodiments having fullysolubilized progesterone and/or estradiol.

As can be seen in Table 16, the solubility of progesterone in CAPMUL®MCM is ˜73 mg/g. Therefore, by suspending 200 mg progesterone in 400 mgof solvent, part of the dose (˜14%) is already dissolved and theremaining is still a suspension. In some aspects and embodiments, it isdesired to minimize the partial solubility of progesterone in theformulation in order to minimize the possibility of recrystallization.

Based on 73 mg/g solubility, the capsule size required to make a capsuleof 50 mg solubilized progesterone would be 685 mg. Therefore, it wasshown that it would be feasible to make a 50 mg progesterone and 2 mgestradiol solubilized formulation. MIGLYOL® had the lowest solubility,but that solvent is unable to dissolve the estradiol, therefore underfurther experiments, it was decided to proceed with the second lowest orCAPMUL® MCM. It has also been found that 2 mg of estradiol may also bedissolved in 685 mg of CAPMUL® MCM.

TABLE 16 Progesterone Solubility Ingredient (mg/g) CAPMUL ® MCM 73.4CAPMUL ® PG8 95 MIGLYOL ® 812 27.8 CAPMUL ® MCM: 86.4 GELUCIRE ® 44/14(9:1) CAPMUL ® MCM: 70.5 GELUCIRE 44/14 (7:3) CAPMUL ® MCM: 57.4GELUCIRE ® 44/14 (6:3)

In addition, it has been found that the solubility of progesterone in asolvent of CAPMUL® MCM in combination with GELUCIRE® 44/14 in a 9:1ratio increases the solubility to approximately 86 mg/g. Therefore, invarious embodiments, progesterone and/or estradiol may be dissolved in aCAPMUL® MCM and GELUCIRE® 44/14 system, wherein the ratio of CAPMUL® MCMto GELUCIRE® 44/14 is 9:1.

TABLE 17 Progesterone Solubility Ingredient (mg/g) CAPMUL ® MCM: 86.4GELUCIRE 44/14 (9:1) CAPMUL ® MCM: 70.5 GELUCIRE ® 44/14 (7:3) CAPMUL ®MCM: 57.4 GELUCIRE ® 44/14 (6:4)

Example 8

In an exemplary embodiment, a capsule is provided containing a fillmaterial having fully solubilized progesterone and estradiol comprising:

TABLE 18 Qty/Capsule Ingredient Mass (mg) % w/w (mg) Progesterone, USP,micronized 50.00 7.14 50.00 Estradiol Hemihydrate, USP 2.03 0.29 2.03CAPMUL ® MCM, NF 82.57 577.97 GELUCIRE ® 44/14, NF 10.0 70.00 TOTAL100.00 700.00

A capsule such as that shown in Table 18 may be manufactured in anysuitable manner. For the purposes of this Example, mixing may befacilitated by an impellor, agitator, or other suitable means. Also forthe purposes of this Example, heating and/or mixing may be performedunder an inert or relatively inert gas atmosphere, such as nitrogen gasN2. Mixing and/or heating for the purposes of this Example may beperformed in any suitable vessel, such as a stainless steel vessel.

For example, CAPMUL® MCM may be heated to between 30° C. to 50° C., morepreferably from 35° C. to 45° C., and more preferably to 40° C.+/−2° C.GELUCIRE® 44/14 may be added to the CAPMUL® MCM and mixed untildissolved. The addition may occur all at once or may occur graduallyover a period of time. Heat may continue to be applied during the mixingof the GELUCIRE® 44/14 and the CAPMUL® MCM.

Heat may be removed from the GELUCIRE® 44/14 and CAPMUL® MCM mixture.Estradiol Hemihydrate may be added to the mixture. The addition mayoccur all at once or may occur gradually over a period of time.Micronized progesterone may then be added to the GELUCIRE® 44/14,CAPMUL® MCM and Estradiol Hemihydrate mixture until dissolved. Theaddition may occur all at once or may occur gradually over a period oftime.

Example 9

In an exemplary embodiment, a capsule is provided containing a fillmaterial having suspended progesterone comprising:

TABLE 19 mg/ Ingredient Capsule % Function Micronized 200.00 30.77Active Progesterone Medium Chain qs qs Carrier Triglyceride (MIGLYOL ®812 or equivalent) Lecithin Liquid 1.63 0.25 Lubricant/ EmulsifierButylated 0.13 0.02 Antioxidant Hydroxytoluene (also referred to as“BHT”)

The above formulation is prepared as follows: MIGLYOL® is heated toabout 45° C. GELUCIRE® 44/14 is added and mixed until dissolved. BHT isadded and mixed until dissolved. Progesterone is suspended and passedthrough a colloid mill. The resultant fill mass can be used forencapsulation.

In an exemplary embodiment, a capsule is provided containing a fillmaterial having partially solubilized progesterone comprising:

TABLE 20 Qty/ Qty/ Amount/ Capsule % Capsule Batch Ingredient (mg) w/w(mg) (kg) Micronized 200.00 33.33 Active 2.0 Progesterone, USPMonoglycerides/ 394.0 65.67 Carrier 3.94 diglycerides/triglycerides ofcaprylic/capric acid (CAPMUL ® MCM) Lauroyl polyoxyl-32- 6.0 1Lubricant/ 0.06 glycerides Emulsifier (GELUCIRE ® 44/14 or equivalent)Total 600.00 mg 100 6.0 kg

For suspensions of progesterone and partially solubilized progesterone,GELUCIRE® 44/14 may be added at 1% to 2% w/w to increase viscosity. Theabove formulation is prepared as follows: CAPMUL® MCM is heated to about65° C. GELUCIRE® 44/14 is added and mixed until dissolved. Heat isremoved. Progesterone is added and the mixture is passed through acolloid mill. The resultant fill mass can be used for encapsulation.

Example 10

In an exemplary embodiment, a capsule is provided containing a fillmaterial having suspended progesterone comprising:

TABLE 21 mg/ Ingredient % Capsule Function Micronized Progesterone 30.77200.00 Active Medium Chain Triglyceride 65.93 428.55 Carrier (MIGLYOL ®812 or equivalent) Lauroyl polyoxyl-32-glycerides 3.00 19.50 Suspending(GELUCIRE ® 44/14 or Agent equivalent) Butylated Hydroxytoluene 0.031.95 Antioxidant Total 100 650

In various embodiments, amounts of MIGLYOL® may be present in a rangefrom about 35-95% by weight; GELUCIRE® 44/14 from about 0.5-30% byweight; and BHT from about 0.01-0.1% by weight.

Example 11

For the purposes of this Example, a particle size analysis is conductedby using the Beckman Device. A sample API comprising micronizedprogesterone in accordance with various embodiments is provided foranalysis.

Approximately 0.01 g of a sample API in accordance with variousembodiments was combined with Coulter 1B and 10 mL of deionized water.Sonication was performed for 15 seconds. The Beckman Device, equippedwith a ULM, performed analysis for 90 seconds. The Beckman Device wasconfigured to use the Fraunhofer optical model. The Beckman Deviceyielded that the sample has an X50 of 4.279 μm, an X75 of 7.442 μm, andan X25 of 1.590 μm. The Beckman Device also yielded that the meanparticle size is 4.975 μm, the median particle size is 4.279 μm, themode particle size is 6.453 μm, and the standard deviation is 3.956 μm.A graph of the particle distribution obtained is shown in FIG. 4.

Example 12

A formulation sample having approximately 200 mg of micronizedprogesterone and 2 mg of estradiol was dispersed with oil. The BeckmanDevice, equipped with a MLM, performed analysis for 60 seconds. TheBeckman Device was configured to use the Fraunhofer optical model. TheBeckman Device yielded that the sample has an X50 of 11.0 μm, an X75 of17.3 μm, and an X25 of 5.3 μm. The Beckman Device also yielded that themean particle size is 11.8 μm, the median particle size is 11.04 μm, themode particle size is 13.6 μm, and the standard deviation is 7.8 μm.

Example 13

In order to increase the solubility of progesterone in the finalsolution, GELUCIRE® 44/14 was added at about 10% w/w.

TABLE 22 Quantitative Formula: Batch Size 10,000 capsules Label Qty/Amount/ Item Claim % Capsule Batch No. Ingredient(s) (mg) w/w (mg)(kg) 1. Progesterone, 50.00 7.14 50.00 0.50 USP, micronized 2. Estradiol2.03 0.29 2.03 0.02 Hemihydrate, USP 3. CAPMUL ® 82.57 577.97 5.78 MCM,NF 4. GELUCIRE ® 10.0 70.00 0.70 44/14, NF Total: 100.00 700.00 7.00

An example of the final formulation is provided in Table 22. Themanufacturing process is as follows. CAPMUL® MCM is heated to 40° C.GELUCIRE® 44/14 is heated to 65° C. and added and mixed until dissolved.Heat is removed. Estradiol is added and mixed until dissolved.Micronized progesterone is then added and mixed until dissolved.

Example 14

In an exemplary embodiment, a capsule is provided containing a fillmaterial having fully solubilized estradiol and partially solubilizedprogesterone comprising:

TABLE 23 Label Qty/ Amount/ Item Claim Capsule Batch No. Ingredient(s)(mg) % w/w (mg) (g) 1. Progesterone, USP, 50.00 25.000 50.00 500.00micronized 2. Estradiol Hemihydrate 0.25 0.129 0.26 2.58 3. CAPMUL ®MCM, 73.371 146.74 1467.42 NF 4. GELUCIRE ® 44/14, 1.500 3.00 30.00 NFTotal: 100.00 200.00 mg 2000.00

The manufacturing process is as follows. CAPMUL® MCM is heated to 65° C.GELUCIRE® 44/14 is added and mixed until dissolved. Heat is removed.Estradiol is added and mixed until dissolved. Micronized progesterone isthen added and dispersed. The mixture is then passed through a colloidmill. The resultant fill mass can be used for encapsulation.

Example 15

In an exemplary embodiment, a capsule is provided containing a fillmaterial having fully solubilized estradiol and partially solubilizedprogesterone comprising:

TABLE 24 Label Qty/ Amount/ Item Claim Capsule Batch No. Ingredient(s)(mg) % w/w (mg) (g) 1. Progesterone, USP, 200.00 33.33 200.0 2000.0micronized 2. Estradiol 2.00 0.35 2.07 20.7 Hemihydrate 3. CAPMUL ® MCM,65.32 391.93 3919.3 NF 4. GELUCIRE ® 1.00 6.0 60.0 44/14, NF Total:100.00 600.0 mg 6000.0

The manufacturing process is as follows. CAPMUL® MCM is heated to 65° C.GELUCIRE® 44/14 is added and mixed until dissolved. Heat is removed.Estradiol is added and mixed until dissolved. Micronized progesterone isthen added and dispersed. The mixture is then passed through a colloidmill. The resultant fill mass can be used for encapsulation.Alternatively, GELUCIRE® 44/14 is heated to 65 C and CAPMUL® MCM isheated to 40 C+/−5 C to achieve mixing of the oil and the surfactantbefore heat is removed; estradiol is added while the mixture is cooling;progesterone is added when the mixture has dropped below about 4° C.;the mixture is then passed through a colloid mill, e.g., three times.

Example 16 Study 352—Progesterone and Estradiol Combination Study UnderFed Conditions

This following study protocol was used to establish bio-availability andbioequivalence parameters for a combination product of the presentdisclosure comprising progesterone (200 mg) and estradiol (2.0 mg) asprepared via the process described in Example 14 and compared to 200 mgof PROMETRIUM® (Catalent Pharmaceuticals, St. Petersburg, Fla. (and 2.0mg of ESTRACE® (estradiol vaginal cream, USP, 0.01%) (Bristol-MyersSquibb Co. Princeton, N.J.), administered to twenty-four (24) normalhealthy, adult human post-menopausal female subjects under fedconditions.

The pharmaceutical formulation of the invention used in these PK studieshad substantially the following formula as shown in Table 25:

TABLE 25 Qty/Capsule Ingredient(s) Amount (% w/w) (mg) Progesterone,33.33 200.00 USP, micronized Estradiol 0.35 2.07 Hemihydrate, USPMicronized CAPMUL 65.32 391.93 MCM, NF, USP GELUCIRE 1.00 6.00 44/14, NFTotal 100.00 600

The Study Design: An open-label, balanced, randomized, two-treatment,two-period, two-sequence, single-dose, two-way crossover study. Thesubjects were housed in the clinical facility from at least 11.00 hourspre-dose to at least 48.00 hours post-dose in each period, with awashout period of at least 14 days between the successive dosing days.

Subjects were fasted for at least about 10.00 hours before being serveda high-fat, high-calorie breakfast, followed by dosing, then followed bya 04.00 hour, post-dose additional period of fasting. Standard mealswere provided at about 04.00, 09.00, 13.00, 25.00, 29.00, 34.00 and38.00 hours post-dose, respectively. Water was restricted at least about01 hour prior to dosing until about 01 hour post-dose (except for watergiven during dosing). At other times, drinking water was provided adlibitum. Subjects were instructed to abstain from consuming caffeineand/or xanthine containing products (i.e. coffee, tea, chocolate, andcaffeine-containing sodas, colas, etc.) for at least about 24.00 hoursprior to dosing and throughout the study, grapefruit and\or its juiceand poppy containing foods for at least about 48.00 hours prior todosing and throughout the study.

Subjects remained seated upright for about the first 04.00 hourspost-dose and only necessary movements were allowed during this period.Thereafter subjects were allowed to ambulate freely during the remainingpart of the study. Subjects were not allowed to lie down (except asdirected by the physician secondary to adverse events) duringrestriction period.

Subjects were instructed not to take any prescription medications within14 days prior to study check in and throughout the study. Subjects wereinstructed not to take any over the counter medicinal products, herbalmedications, etc. within 7 days prior to study check-in and throughoutthe study.

After overnight fasting of at least about 10.00 hours, a high-fathigh-calorie breakfast was served about 30 minutes prior toadministration of investigational product(s). All subjects were requiredto consume their entire breakfast within about 30 minutes of it beingserved, a single dose of either test product (T) of Progesterone 200 mg& Estradiol 2 mg tablets or the reference product (R) PROMETRIUM®(Progesterone) soft gel Capsule 200 mg and ESTRACE® (Estradiol) Tablets2 mg (according to the randomization schedule) were administered withabout 240 mL of water under fed condition, at ambient temperature ineach period in sitting posture. A thorough mouth check was done toassess the compliance to dosing.

All dosed study subjects were assessed for laboratory tests at the endof the study or as applicable.

In each period, twenty-three (23) blood samples were collected. Thepre-dose (10 mL) blood samples at −01.00, −00.50, 00.00 hours and thepost-dose blood samples (08 mL each) were collected at 00.25, 00.50,00.67, 00.83, 01.00, 01.33, 01.67, 02.00, 0.50, 03.00, 04.00, 05.00,06.00, 07.00, 08.00, 10.00, 12.00, 18.00, 24.00 and 48.00 hours inlabeled K2EDTA—vacutainers via an indwelling cannula placed in one ofthe forearm veins of the subjects. Each intravenous indwelling cannulawas kept in situ as long as possible by injecting about 0.5 mL of 10IU/mL of heparin in normal saline solution to maintain the cannula forcollection of the post-dose samples. In such cases, blood samples werecollected after discarding the first 0.5 mL of heparin containing blood.Each cannula was removed after the 24.00 hour sample was drawn orearlier or if blocked.

At the end of the study, the samples were transferred to thebio-analytical facility in a box containing sufficient dry ice tomaintain the integrity of the samples. These samples were stored at atemperature of −70° C.±20° C. in the bio-analytical facility untilanalysis.

Progesterone (corrected and uncorrected), estradiol (unconjugated),total estrone, and estrone sulfate in plasma samples is assayed using avalidated LC-MS/MS method.

The pharmacokinetic parameters C_(max), AUC_((0-t)) and AUC_((0-∞)) werecalculated on data obtained from 24 subjects for the test product andreference product. In general, bioavailability of progesterone andestradiol were similar but bioequivalence was not established.

Corrected pharmacokinetic profile summaries are presented in Table 26,below, for progesterone.

TABLE 26 Summary of Primary Pharmacokinetic Profile of Test Product (T)versus Reference Product (R) for Progesterone (Corrected) ArithmeticMean ± Standard Pharmaco- Geometric Mean* Deviation kinetic TestReference Test Product Reference Parameter Product (T) Product (R) (T)Product(R) C_(max) 47.0 43.0 81.0 ± 82.8 117.7 ± 173.7 AUC_(0-t) 107.697.8 163.9 ± 136.5 191.1 ± 241.7 AUG_(0-∞) 110.7 110.0 173.5 ± 143.0207.1 ± 250.3 *Estimate of Least Square Mean used to calculate GeometricMean

Example 17 Study 351—Progesterone and Estradiol Combination Study underFasting Conditions

Fasted studies using the above protocol and test and reference productswere also conducted. However, rather than the high-fat meal prior toadministration of the test and reference drug, each subject fasted for aperiod of at least twelve (12) hours prior to dose administration.

The pharmacokinetic parameters C_(max), AUC_((0-t)) and AUC_((0-∞)) werecalculated on data obtained from 23 subjects under fasting conditionsfor the test product and reference product. In general, bioavailabilityof progesterone and estradiol were similar but bioequivalence was notestablished.

Corrected pharmacokinetic profile summaries are presented in Table 27,below, for progesterone.

TABLE 27 Summary of Primary Pharmacokinetic Profile of Test Product (T)versus Reference Product (R) for Progesterone (Corrected) ArithmeticMean ± Standard Pharmaco- Geometric Mean* Deviation kinetic TestReference Test Reference Parameter Product (T) Product (R) Product (T)Product(R) C_(max) 2.3 3.0  2.9 ± 2.3 3.9 ± 3.4 AUC_(0-t) 8.4 10.9 11.2± 8.7 14.5 ± 11.0 AUG_(0-∞) 12.9 17.2 15.1 ± 9.0 19.6 ± 10.2 *Estimateof Least Square Mean used to calculate Geometric Mean

The data indicate good (i.e., low) inter-patient and intra-patientvariability relative to PROMETRIUM.

Example 18

Methods of manufacture in accordance with various embodiments are shownin FIGS. 1-3. With reference to FIG. 1, method of fill material 100 isshown. Step 102 comprises heating an oily vehicle carrier to 40° C.±5°C. Heating may be accomplished through any suitable means. The heatingmay be performed in any suitable vessel, such as a stainless steelvessel. The oily vehicle may be any oily vehicle described herein, forexample, CAPMUL® MCM.

Step 104 comprises mixing GELUCIRE® 44/14 with the oily vehicle. Mixingmay be facilitated by an impellor, agitator, or other suitable means.Step 102 may be performed under an inert or relatively inert gasatmosphere, such as nitrogen gas N2. Mixing may be performed in anysuitable vessel, such as a stainless steel vessel.

Step 106 comprises mixing estradiol into the mixture of the oily vehicleand GELUCIRE® 44/14. Mixing may occur in a steel tank or vat. Mixing maybe facilitated by an impellor, agitator, or other suitable means. Step106 may be performed under an inert or relatively inert gas atmosphere,such as nitrogen gas N2.

Step 108 comprises cooling to room temperature. Cooling may be allowedto occur without intervention or cooling may be aided by application ofa cooling system.

Step 110 comprises mixing micronized progesterone into the mixture ofoily vehicle, estradiol and GELUCIRE® 44/14. Mixing may occur in a steeltank or vat. Mixing may be facilitated by an impellor, agitator, orother suitable means. Step 110 may be performed under an inert orrelatively inert gas atmosphere, such as nitrogen gas N2. Step 112comprises degasing. The resulting mixture from step 112 may comprise afill material suitable for production into a softgel capsule.

With reference to FIG. 2, softgel capsule, i.e. gel mass, production 200is shown. Step 202 comprises mixing glyercin with water. The water usedin step 202 may be purified by any suitable means, such as reverseosmosis, ozonation, filtration (e.g., through a carbon column) or thelike. Mixing may be facilitated by an impellor, agitator, or othersuitable means. Step 202 may be performed under an inert or relativelyinert gas atmosphere, such as nitrogen gas N2. Heating may be performeduntil the temperature reaches 80° C.±5° C.

Step 204 comprises the addition of gelatin to the glycerin watermixture. Mixing may be facilitated by an impellor, agitator, or othersuitable means. Step 204 may be performed under an inert or relativelyinert gas atmosphere, such as nitrogen gas N2. A vacuum may be drawn instep 204 to de-aerate.

Step 206 comprises addition of a coloring agent such as a dye. Acoloring agent may comprise products sold under the trademark OPATINT orother suitable agent. Step 206 may be performed under an inert orrelatively inert gas atmosphere, such as nitrogen gas N2. Step 208comprises degasing. The resulting mixture from step 208 may comprise agel capsule material suitable for use as a gel capsule in production ofa softgel capsule.

With reference to FIG. 3, softgel capsule assembly process 300 is shown.Step 302 comprises heating the fill material. The fill material may beheated to any suitable temperature. In various embodiments, the fillmaterial is heated to 30° C.+/−3° C. Fill material may be heated in afill hopper. A fill hopper may comprise a device configured to hold avolume of the fill material and/or to dispense the fill material incontrolled volumes.

Step 304 comprises filling a gel mass. A gel mass may be taken from thegel capsule material produced in step 208 of FIG. 2. Filling may beperformed by injecting, placing, or otherwise disposing the fillmaterial within a volume defined by the gel capsule material. Thefilling may occur in an encapsulator. The spreader boxes may be atemperature of 55° C.+/−10° C. The wedge temperature may be 38° C.+/−3°C. The drum cooling temperature may be 4° C.+/−2° C. The encapsulatormay be lubricated using MIGLYOL® 812 or other suitable lubricant. Step304 thus produces one or more softgel capsules. Filling may compriseproducing a ribbon of thickness 0.85 mm±0.05 mm using spreader boxknobs. The fill material may be injected into the gel to produce a fillweight having target weight±5% (i.e., 650±33 mg and 325±16.3 mg).

Step 306 comprises drying the softgel capsules. Drying may be performedin a tumble dryer, tray dryer, or combinations thereof. For example,drying may be performed in a tumble drying basket for between about 10minutes and about 120 minutes. Drying may continue in a drying room forabout 24 hours to about 72 hours. Step 308 may comprise inspectionand/or polishing. Polishing may be performed with isopropyl alcohol.Step 310 may comprise packaging. Packaging may be accomplished throughany suitable means. Packaging may comprise packing softgel capsules intoa blister pack, bottle, box, pouch, or other acceptable packaging.

Example 19 I. Solubility of Estradiol in Soy Bean Oil, Peanut Oil, andSafflower Oil

Data was obtained visually by making the mixtures described below,sonicating the mixtures, and then seeing if a clear solution resulted.If a clear solution was achieved, it was an indication of solubility atthe level studied.

Procedures and Results:

Step 1.

0.3% of Estradiol suspension in each oil was prepared by adding 30 mgEstradiol to solvent and QS to 10 g. Samples were mixed on vortex for 2hours, heated at 50° C. for 30 minutes and then mixed for 1 hour more.All samples were still in suspension form.

Step 2.

Each sample was diluted to 0.24% (by adding 2.5 g more oil) and mixedfor 2 hours and heated at 50° C. for 30 min and mixed again for onehour. All the samples were still cloudy. Samples were kept at roomtemperature overnight to see if they precipitate or if un-dissolved APIsettles out. After 20 hours at room temperature, it was observed thatall samples still had un-dissolved API.

Step 3.

Each sample was diluted to 0.2% (by adding 2.5 g more oil) and mixed 2for hours and heated at 50° C. for 30 min and mixed again for one hour.All the samples were still slightly cloudy, indicating that theestradiol was not completely dissolved.

TABLE 28 Estradiol Solubility Estradiol Solubility Ingredient (mg/g) (%w/w) Peanut Oil <2 <0.2 Safflower Oil <2 <0.2 Soy Bean Oil <2 <0.2

The solubility of estradiol in all three oils was less than 2 mg/g (0.2%w/w). This level of solubility is significantly below the solubilitythat the present inventors have discovered can be achieved in otheroils, e.g., medium chain fatty acid esters, such as themono/diglycerides, propylene glycol esters, and polyethylene glycolesters discussed above.

In sum, if no heat is used to dissolve estradiol in safflower oil, itwill not go into solution. Given that the estradiol did not dissolve at50° C., oils such as safflower oil will not be useful in the methods ofthe invention using medium chain fatty acid esters as describedhereinabove.

II. Solubility in Safflower Oil

In a separate experiment, 50 g of safflower oil was heated to 85-88° C.and 60 mg estradiol was added, mixed until fully dissolved (1 hr), andallowed to cool to room temperature. The solubility achieved was 1.0mg/ml. Addition of progesterone to a sample of the estradiol solutiondid not affect the solubility of estradiol.

Unsaturated fats are prone to oxidation, i.e., rancidity. Peroxides areintermediates formed during oxidation and the Peroxide Value is anindicator of extent of oxidation. The US Pharmacopeia specification forPeroxide Value of safflower oil is 10 max. Heating the oil, e.g., to 85°C., has been shown to increase the Peroxide Value. In contrast, mediumchain fatty acid glycols, such as CAPMUL® MCM and MYGLYOL® 812, whichcomprise saturated C8-C10 fatty acid esters, have much lower PeroxideValues, e.g., on the order of 1 or less.

Example 20 Dissolution

Dissolution studies were performed using a formulation of this inventioncomparing the dissolution of progesterone to the dissolution ofPROMETRIUM® and comparing the dissolution of estradiol to thedissolution of ESTRACE®. In one study, a formulation of the invention incapsules comprising 200 mg of progesterone and 2 mg estradiol was used.In a second study, a formulation of the invention in capsules comprising50 mg of progesterone and 2 mg estradiol was used. The two formulationscomprised:

The dissolution study was performed using a USP dissolution apparatus(reciprocating cylinder) (“USP Apparatus 3”). The apparatus was set to30 dips per minute. 250 mL of a solution of 0.1N HCl with 3% sodiumlauryl sulfate was used at 37° C.

In both studies, progesterone was dissolved faster, and with smallerstandard deviations, from the capsules of the invention than fromPROMETRIUM®. Dissolution of estradiol was comparable but marginallyslower from the capsules of the invention than from ESTRACE®. Forillustrative purposes, a graph showing progesterone dissolution from the200 mg progesterone capsule of the invention and from PROMETRIUM® isattached as FIG. 5.

Both capsules of the invention were stable on storage in white HDPEbottles. Positive stability data were obtained with the 200 mgprogesterone formulation over 6 months (>6 months data unavailable) andwith the 50 mg progesterone formulation over 3 months (>3 months dataunavailable).

Example 21 Study 459—Combination Study under FedConditions—Pharmacokinetics of the First Combination17β-Estradiol/Progesterone Capsule in Clinical Development for HormoneTherapy

The objective of this study was to evaluate the pharmacokinetic and oralbioavailability of a combination capsule of 17β-estradiol/progesteronein comparison to co-administration of the individual products ESTRACE®and PROMETRIUM®.

Subjects and Study Design:

An open label, balanced, randomized, single-dose, 2-treatment, 3-period,3-sequence, crossover, partial-replicate, reference-scaled, oral,relative bioavailability study compared the bioavailability of aninvestigational 2-mg 17β-estradiol/200-mg progesterone combinationcapsule, without peanut oil (formulated in a manner similar to that setforth in Table 24), with that of co-administered 200-mg PROMETRIUM®(progesterone) and 2-mg ESTRACE® (17β-estradiol) tablets in healthypostmenopausal women aged 40-65 yrs (N=66). Key inclusion criteria forsubjects included a BMI 18.50 to 29.99 kg/m² who were nonsmokers orex-smokers (no smoking in the last 3 months). Key exclusion criteria forsubjects included consuming grapefruit juice or poppy-containing foodswithin 48 hours before and throughout the study, use of any hormonalagent within 14 days before the study, and use of menopausal hormonetherapy within 6 months before dosing.

Patients were randomly assigned sequentially to 1 of 3 dosing sequencesof the same dose of the combination capsule (Test, T) and referenceproducts (Reference, R): TRR, RTR, or RRT. 66 subjects were randomizedand 62 (94.0%) completed the study (FIG. 6). Subjects had a mean age of49.5±5.6 years (range 40 to 64) and a mean BMI of 24.8±3.1 kg/m² (range18.7-29.9).

After consuming a high-fat, high-calorie breakfast, each woman receiveda single dose of the combination (Test) capsule in 1 period of the studyand single doses of the co-administered products (Reference) in each ofthe 2 remaining periods. Plasma was collected pre-dose and at specifiedintervals over 48 h after dosing to determine progesterone, free(unconjugated) estradiol, and free and total (conjugated+free) estroneconcentrations. The primary (C_(max), AUC_(0-t), and AUC_(0-∞)) andsecondary (t_(max), t_(1/2), and K_(e)) PK parameters for each analytewere determined for each subject during each period by non-compartmentanalyses using baseline-adjusted concentrations. Statistical analyseswere conducted using the Scaled Average Bioequivalence (BE) method forhighly variable drugs to determine whether the combination Test capsulehad progesterone and estradiol bioavailability similar to that of theco-administered Reference products. This method is applicable when thewithin-subject coefficient of variation (CV) for the reference productis ≧30%. For any PK parameter with CV<30%, the BE method based on 90% CImust be used.

Results: All AUC and C_(max) parameters met BE criteria for allanalytes, except C_(max) for total estrone (Table 29). The extent ofestradiol and progesterone absorption for the Test capsule appeared tobe similar to that for the ESTRACE® and PROMETRIUM® tablets,respectively (Table 29), while the rate of estradiol absorption for theTest capsule appeared to be faster than that for ESTRACE®, respectively(Table 30). Semilogarithmic plots of AUC over time for each analyte arepresented in FIGS. 7 and 8. Pharmacokinetic data (C_(max), AUC_((0-t)),and AUC_((0-∞))) for progesterone, estradiol, free estrone, and totalestrone is presented in Tables 31-34. For Tables 31-34, “Test Product(T)” refers to the progesterone+estradiol pharmaceutical composition,while “Reference product (R1)” and “Reference product (R2)” refer toco-administered PROMETRIUM® (progesterone) and ESTRACE® (estradiol).

The bioequivalent outcomes for progesterone should allow the Testcapsule to bridge to the safety data for PROMETRIUM®. A bridge to thesafety data for the ESTRACE® tablet should also be viewed as having beenestablished because the only parameter for which the Test capsule failedto meet BE criteria was the C_(max) for just 1 estradiol-related analyte(total estrone). The extent of estradiol absorption, reflecting thetotal amount of estradiol delivered by the Test capsule, is the mostcritical safety measure.

TABLE 29 Scaled Average Bioequivalence Analyses for Each AnalyteTest-to-Ref 95% Upper Meets BE Analyte/Parameter Ratio CV % ConfidenceBound Criteria† Progesterone AUC_((0-t)) 1.05 122.2 −0.5422 YesAUC_((0-inf)) 0.94 116.4 −0.4941 Yes C_(max) 1.16 173.7 −0.7850 YesUnconjugated estradiol AUC_((0-t)) 0.93 42.6 −0.0888 Yes AUC_((0-inf))0.83 47.4 −0.0625 Yes C_(max) 0.88 35.4 −0.0399 Yes Test-to-Ref 90%Confidence Meets BE Analyte/Parameter Ratio CV % Interval CriteriaUnconjugated estrone AUC_((0-t)) 0.89 18.0 0.848-0.938 Yes AUC_((0-inf))0.88 26.3 0.834-0.933 Yes C_(max) 0.93 23.3 0.873-0.991 Yes Totalestrone AUC_((0-t)) 1.06 29.7 0.982-1.115 Yes AUC_((0-inf)) 1.06 29.70.985-1.114 Yes C_(max) 1.75 35.9 0.3438* No *95% Upper Confidence Bound†Scaled Average Bioequivalence requires Test-to-Reference ratio between0.800 and 1.250 and the 95% upper confidence bound on the linearizedstatistic is ≦0. Unscaled Average Bioequivalence requires that the 90%confidence interval on the Test-to-Reference ratio is entirely within0.800 and 1.250. BE = Bioequivalence; CV % = coefficient of variance.

TABLE 30 T_(max) for Each Analyte Analyte/Parameter Test Reference 1Reference 2 Progesterone 3.00 (0.83-10.0) 3.00 (1.00-12.0) 4.00(0.67-18.0) Unconjugated estradiol 9.00 (0.50-36.0) 10.0 (0.50-35.1)10.0 (0.25-36.6) Unconjugated estrone* 5.50 (0.83-36.0) 8.00 (1.67-18.0)10.0 (1.67-18.0)

TABLE 31 Summary of Pharmacokinetic Parameters of Test Product (T)versus Reference Product (R1, R2) for Progesterone (Corrected) ReferenceReference PK Parameter N Test Product (T) N Product (R1) N Product (R2)C_(max) (ng/mL) 62  89.2222 ± 149.7309 62  72.7228 ± 101.8885 62 69.7590± 87.0777 Arithmetic Mean ± SD C_(max) (ng/mL) 62 35.0996 62 30.6904 6229.7178 Geometric Mean AUC_((0-t)) 62 120.0869 ± 164.1385 62 125.9406 ±152.3483 62 111.5867 ± 113.3200 (ng · hr/mL) Arithmetic Mean ± SDAUC_((0-t)) 62 63.3952 62 61.5312 62 58.5421 (ng · hr/mL) Geometric MeanAUC_((0-∞)) 57 131.3817 ± 172.4806 57 142.1332 ± 160.4853 56 126.6006 ±117.2665 (ng · hr/mL) Arithmetic Mean ± SD AUC_((0-∞)) 57 72.1098 5779.9008 56 75.7201 (ng · hr/mL) Geometric Mean

TABLE 32 Summary of Pharmacokinetic Parameters of Test Product (T)versus Reference Product (R1, R2) for Estradiol (Corrected) ReferenceReference PK Parameter N Test Product (T) N Product (R1) N Product (R2)C_(max) (pg/mL) 62 64.7902 ± 50.9833 62 69.1286 ± 33.0484 62 73.4236 ±43.4077 Arithmetic Mean ± SD C_(max) (pg/mL) 62  56.1068 62  62.2189 62 64.5362 Geometric Mean AUC_((0-t)) 62 1403.7333 ± 763.8136  621508.2206 ± 876.7390  62 1658.2502 ± 976.5556  (pg · hr/mL) ArithmeticMean ± SD AUC_((0-t)) 62 1224.2031 62 1239.6990 62 1413.7331 (pg ·hr/mL) Geometric Mean AUC_((0-∞)) 60 2459.4394 ± 4498.2737 60 2842.8805± 4582.6502 57 2110.9591 ± 1175.3995 (pg · hr/mL) Arithmetic Mean ± SDAUC_((0-∞)) 60 1658.0281 60 1879.6716 57 1796.6988 (pg · hr/mL)Geometric Mean

TABLE 33 Summary of Pharmacokinetic Parameters of Test Product (T)versus Reference Product (R1, R2) for Total Estrone (Corrected)Reference Reference PK Parameter N Test Product (T) N Product (R1) NProduct (R2) C_(max) (pg/mL) 61 35.4289 ± 17.0856 61 19.8716 ± 7.4485 6119.9048 ± 8.0288 Arithmetic Mean ± SD C_(max) (pg/mL) 61  31.9856 61 18.3037 61  18.4035 Geometric Mean AUC_((0-t)) 61 201.7524 ± 94.2081 61 182.7729 ± 88.8386 61 199.8295 ± 94.9392 (pg · hr/mL) Arithmetic Mean± SD AUC_((0-t)) 61 182.7135 61 165.3741 61 182.1279 (pg · hr/mL)Geometric Mean AUC_((0-∞)) 61 213.2402 ± 104.6011 60  193.6387 ±100.5831 56 203.0289 ± 81.4884 (pg · hr/mL) Arithmetic Mean ± SDAUC_((0-∞)) 61 191.4769 60 173.4694 56 187.8867 (pg · hr/mL) GeometricMean

TABLE 34 Summary of Pharmacokinetic Parameters of Test Product (T)versus Reference Product (R1, R2) for Estrone Sulfate ReferenceReference PK Parameter N Test Product (T) N Product (R1) N Product (R2)C_(max) (pg/mL) 62 426.5492 ± 179.3303 62  455.5107 ± 189.4486 62 467.2302 ± 207.4373 Arithmetic Mean ± SD C_(max) (pg/mL) 62  391.659162  416.8218 62  425.6676 Geometric Mean AUC_((0-t)) 62 9096.0907 ±4377.2730 62 10156.0282 ± 5140.5831 62 10507.3557 ± 5183.1289 (pg ·hr/mL) Arithmetic Mean ± SD AUC_((0-t)) 62 8043.8229 62 8872.7467 629204.9744 (pg · hr/mL) Geometric Mean AUC_((0-∞)) 61 11994.9695 ±6678.5468  62 13445.9048 ± 8699.4068 62 14066.2362 ± 7563.2370 (pg ·hr/mL) Arithmetic Mean ± SD AUC_((0-∞)) 61 10264.7576  62 11273.4294  6211936.6967  (pg · hr/mL) Geometric Mean

For the test product pharmaceutical formulation, pharmacokinetic dataranges for AUC and C_(max) are presented in Table 35 below.

TABLE 35 pK Ranges for the Test Product (T) Pharmaceutical FormulationC_(max) AUC_((0-t)) AUC_((0-∞)) Progesterone  71 ng/mL to   96 ng ·hr/mL to  105 ng · hr/mL to 112 ng/mL  150 ng · hr/mL  164 ng · hr/mLEstradiol  52 pg/mL to  1123 pg · hr/mL to  1968 pg · hr/mL to  81 pg/mL 1755 pg · hr/mL  3075 pg · hr/mL Estrone sulfate 341 pg/mL to  7277 pg· hr/mL to  9596 pg · hr/mL to 533 pg/mL 11370 pg · hr/mL 14994 pg ·hr/mL Total estrone  28 pg/mL to  161 pg · hr/mL to  171 pg · hr/mL to 44 pg/mL  252 pg · hr/mL  267 pg · hr/mL

CONCLUSION

The combination 17β-estradiol/progesterone capsule demonstrated similarbioavailability of its constituents to their individual respectivereferences of ESTRACE® and PROMETRIUM®, when given together under fedconditions. This new capsule could represent an interesting developmentin hormone therapy, as no approved hormone therapy to date has been ableto 1) combine natural progesterone with 17β-estradiol as an oralformulation, and 2) provide progesterone without peanut oil, a knownallergen. The efficacy and safety of this new capsule combining17β-estradiol with progesterone will be evaluated in phase 3 clinicaltrials.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present disclosurewithout departing from the spirit or scope of the disclosure. Thus, itis intended that the present disclosure cover the modifications andvariations of this disclosure provided they come within the scope of theappended claims and their equivalents.

Likewise, numerous characteristics and advantages have been set forth inthe preceding description, including various alternatives together withdetails of the structure and function of the devices and/or methods.This disclosure is intended as illustrative only and as such is notintended to be exhaustive. It will be evident to those skilled in theart that various modifications may be made, especially in matters ofstructure, materials, elements, components, shape, size and arrangementof parts including combinations within the principles of the disclosure,to the full extent indicated by the broad general meaning of the termsin which the appended claims are expressed. To the extent that thesevarious modifications do not depart from the spirit and scope of theappended claims, they are intended to be encompassed therein.

What is claimed is:
 1. A pharmaceutical formulation for co-administeringestradiol and progesterone to a mammal in need thereof, comprisingsolubilized estradiol, suspended progesterone, and a medium chain(C6-C12) oil, wherein when administered to a human subject, theformulation produces: (a) one or more progesterone-related parametersselected from: (i) an area under the curve (AUC)_((0-t)) forprogesterone that is from 96 ng·hr/ml to 150 ng·hr/ml; (ii) anAUC_((0-∞)) for progesterone that is from 105 ng·hr/ml to 164 ng·hr/ml;and (iii) a C_(max) for progesterone that is from 71 ng/ml to 112 ng/ml;and (b) one or more estrogen-related parameters selected from: (i) anAUC_((0-t)) for estradiol that is from 1123 pg·hr/ml to 1755 pg·hr/ml;(ii) an AUC_((0-∞)) for estradiol that is from 1968 pg·hr/ml to 3075pg·hr/ml; and (iii) a C_(max) for unconjugated estradiol that is from 52pg/ml to 81 pg/ml.
 2. The pharmaceutical formulation of claim 1, whereinadministration of the formulation to the subject produces both anAUC_((0-t)) for progesterone that is from 96 ng·hr/ml to 150 ng·hr/mland a C_(max) for progesterone that is from 71 ng/ml to 112 ng/ml. 3.The pharmaceutical formulation of claim 1, wherein administration of theformulation to the subject produces both an AUC_((0-t)) for unconjugatedestradiol that is from 1123 pg·hr/ml to 1755 pg·hr/ml and a C_(max) forunconjugated estradiol that is from 52 pg/ml to 81 pg/ml.
 4. Thepharmaceutical formulation of claim 1, wherein when administered to ahuman subject, the formulation further produces one or more of thefollowing: (i) an AUC_((0-t)) for estrone sulfate that is from 7277pg·hr/ml to 11370 pg·hr/ml; (ii) an AUC_((0-∞)) for estrone sulfate thatis from 9596 pg·hr/ml to 14994 pg·hr/ml; or (iii) a C_(max) for estronesulfate that is from 341 pg/ml to 533 pg/ml.
 5. The pharmaceuticalformulation of claim 4, wherein administration of the formulation to thesubject produces both an AUC_((0-t)) for estrone sulfate that is from7277 pg·hr/ml to 11370 pg·hr/ml and a C_(max) for estrone sulfate thatis from 341 pg/ml to 533 pg/ml.
 6. The pharmaceutical formulation ofclaim 1, wherein when administered to a human subject, the formulationfurther produces one or more of the following: (i) an AUC_((0-t)) fortotal estrone that is from 161 pg·hr/ml to 252 pg·hr/ml; (ii) anAUC_((0-∞)) for total estrone that is from 171 pg·hr/ml to 267 pg·hr/ml;or (iii) a C_(max) for total estrone that is from 28 pg/ml to 44 pg/ml.7. The pharmaceutical formulation of claim 6, wherein administration ofthe formulation to the subject produces both an AUC_((0-t)) for totalestrone that is from 161 pg·hr/ml to 252 pg·hr/ml and a C_(max) fortotal estrone that is from 28 pg/ml to 44 pg/ml.
 8. A method of treatinga subject having one or more symptoms of estrogen deficiency, the methodcomprising administering to the subject an effective amount of thepharmaceutical formulation of claim
 1. 9. The method of claim 8, whereinthe subject is female.
 10. The method of claim 8, wherein the subject isa woman having a uterus.